Pulmonary delivery for bioconjugation

ABSTRACT

Methods of and compositions for pulmonary delivery of therapeutic agents which are capable of forming covalent bonds with a site of interest or which have formed a covalent bond with a pulmonary solution protein are disclosed. Therapeutic agents useful in the invention include wound healing agents, antibiotics, anti-inflammatories, anti-oxidants, anti-proliferatives, immunosupressants, anti-infective and anti-cancer agents.

FIELD OF THE INVENTION

[0001] This invention relates to the field of therapeutic and diagnosticagents in medicine. In particular, this invention relates to the fieldof delivery, in particular, pulmonary delivery, of therapeutic anddiagnostic agents wherein the agents are capable of covalently bondingto a site of interest in vivo, to provide increased bioavailability andpharmacodynamic duration of therapeutic and diagnostic benefit for thegiven agent.

BACKGROUND OF THE INVENTION

[0002] Peptide and protein drugs are being used increasingly in majorresearch and development programs in the pharmaceutical industry and arealso an important class of therapeutic agents due to advances in geneticengineering and biotechnology. Systemic delivery of these macromoleculardrugs and other therapeutic and diagnostic agents, however, has beenlimited to the parenteral route largely because of their extensivepresystemic elimination when taken orally. Faced with this dilemmaconcerning the systemic delivery of these macromolecules with theirunique conformational complexity for therapeutic activity,pharmaceutical scientists are continually evaluating the potential ofvarious non-oral routes of administration as alternatives.

[0003] Despite the tremendous efforts that have been devoted to thisproblem, only limited success has been achieved—mostly with smallpeptides. An alternative, non-invasive means for systemic delivery oftherapeutic and diagnostic agents is via the pulmonary system. Deliveryvia the pulmonary system is advantageous because the lungs provide alarge but extremely thin absorptive mucosal membrane for increasedabsorption and delivery to the blood stream. However, pulmonary deliveryof therapeutic and diagnostic agents is complicated by the complexity ofthe anatomic structure of the human respiratory system; the effect ofrespiration on drug deposition and an instability of the drugs resultingfrom degradation in either the lungs or plasma.

[0004] There is thus a need to improve and enhance delivery oftherapeutic and diagnostic agents, especially pulmonary delivery of oftherapeutic and diagnostic agents through increasing the stability andblood absorption of the agents.

SUMMARY OF THE INVENTION

[0005] In order to meet these needs, the present invention is directedto therapeutic and diagnostic agents capable of forming covalent bondsto blood and pulmonary fluid proteins or other components ex vivo or invivo. The therapeutic agents of this invention have a long duration ofaction for the management of disease. The invention relates to ex vivoand in vivo. bioconjugation of therapeutic agents to protein (e.g.albumin), as well as an intrapulmonary in vivo bioconjugation oftherapeutic agents to endogenous pulmonary fluid proteins or othercomponents to dramatically increase the half life of the therapeuticagents and avoid the need for parenteral administration.

[0006] The present invention reflects the ability to bioconjugateselected therapeutic agents to blood and pulmonary pulmonary fluidproteins, including albumin, for processing as a particulate forintrapulmonary drug delivery. The pulmonary fluid protein conjugate istargeted to provide a stable drug substance that retains biologicalactivity for prolonged periods of time. This invention providesprolonged local retention of therapeutic agent activity in the airwaysfor use with selected therapeutic agents in managing pulmonary disease.

[0007] The invention is further directed to methods of facilitatingsystemic drug delivery of protein-therapeutic agent bioconjugates and toagents capable of forming bioconjugates to protein in vivo via pulmonarydelivery with subsequent transcytosis across the alveolar and pulmonarymucosa. The invention is further directed to methods of facilitatingsystemic drug delivery of protein-therapeutic agent bioconjugates viapulmonary delivery of agents capable of forming bioconjugates in vivo,the agents crossing the epithelium of the alveolar or pulmonary mucosa,either through diffusion or receptor-mediated transport, to conjugatewith blood proteins. The methods of this invention result in longacting, systemic therapeutics that are stabilized by ex vivo or in vivoconjugation to pulmonary fluid proteins and/or blood proteins.

[0008] This invention is further directed to site-specific andprotein-specific bioconjugation of a therapeutic agent to albumin.Albumin possesses a unique nucleophilic moiety, specifically, the thiolfunctionality on cysteine 34 that is capable of undergoing anucleophilic attack on electrophile present on a therapeutic agentmodified according to the invention. This selective covalent bondingenables bioconjugation to extracellular as well as intracellular albuminfor prolonged retention and bioavailabilty of the therapeutic agent.

[0009] This invention is further directed to the use of reactivechemistries including: N-hydroxy sulfosuccinimide,maleimide-benzoyl-succinimide, gamma-maleimido-butyryloxy succinimideester, maleimidopropionic acid, isocyanate, thiolester, thionocarboxylicacid ester, imino ester, and carbodiimide anhydride. Maleimidopropionicacid is the preferred reactive chemistry, but the invention alsocontemplates the selection of the above and like reactive chemistries asan electrophilic moeity for bioconjugations with albumin or otherproteins.

[0010] This invention is further directed to the use of a compositionfor the manufacture of a medicament where the composition comprises aderivative of an antihistamine and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananhistamine effect.

[0011] The modified antihistamine may be cetirizine, loratidine andanalogs thereof.

[0012] This invention is further directed to the use of a compositionfor for the manufacture of a medicament where the composition comprisesa derivative of an anti-angina agent and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananti-angina effect.

[0013] The modifed anti-angina agent may be tirofiban or analogsthereof.

[0014] This invention is further directed to the use of a compositionfor the manufacture of a medicament where the composition comprises aderivative of an anti-hypertensive agent and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananti-hypertensive effect.

[0015] The anti-hypetensive agent may be enalapril or analogs thereof.

[0016] This invention is further directed to the use of a compositionfor the manufacture of a medicament where the composition comprising aderivative of an anti-arrhythmic agent and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananti-arrhythmic effect.

[0017] The anti-arrhythmic agent may be capobenic acid or analogsthereof.

[0018] This invention is further directed to the use of a compositionfor the manufacture of a medicament where the composition comprising aderivative of an anti-depressant agent and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananti-depressan effect.

[0019] The anti-depressant agent may be fluoxetine or analogs thereof.

[0020] This invention is further directed to the use of a compositionfor the manufacture of a medicament said composition comprising aderivative of a bronchodilator and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide abronchodilation effect.

[0021] The bronchodilator may be theobromineacetamine or analogsthereof.

[0022] This invention is further directed to the use of a compositionfor the manufacture of a medicament said composition comprising aderivative of an opioid and analogs thereof, wherein the derivativeincludes a reactive functional group which reacts with amino groups,hydroxyl groups, or thiol groups on blood components to form stablecovalent bonds, said reactive functional group being selected fromN-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide groupfor use in the treatment of the human body to provide an analgesiceffect.

[0023] The opioid may be fentanyl or analogs thereof.

[0024] This invention is further directed to the use of a compositionfor the manufacture of a medicament said composition comprising aderivative of an anti-inflammatory agent and analogs thereof, whereinthe derivative includes a reactive functional group which reacts withamino groups, hydroxyl groups, or thiol groups on blood components toform stable covalent bonds, said reactive functional group beingselected from N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and amaleimide group for use in the treatment of the human body to provide ananti-inflammatory effect.

[0025] The anti-inflammatory agent may be loxoprofen or analogs thereof.

[0026] This invention is further directed to the use of a compositionfor the manufacture of a medicament where the composition comprising aderivative of an anti-thyroid deficiency agent and analogs thereof,wherein the derivative includes a reactive functional group which reactswith amino groups, hydroxyl groups, or thiol groups on blood componentsto form stable covalent bonds, said reactive functional group beingselected from N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and amaleimide group for use in the treatment of the human body to provide ananti-thyroid deficiency effect.

[0027] the anti-thyroid deficiency agent may be thyroxin or analogsthereof.

[0028] This invention is further directed to composition comprising oneor more compounds selected from the group consisting of2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide;11-(N-maleimidopropionyl-4-piperidylidene)-8-chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-pyridine;N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropionilamide;Maleimidopropynamyl-ε-(3,4,5-trimethoxybenz-amido)-caproicamide;Maleimidopropionamyl-1-theobromineacetamide;Maleimidopropamyl2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamideN-maleimidopropionyl-N-methyl-3-(p-trifluoromethylphenoxy)-3-phenylpropylamine;4-anilino-1-(2-phenethyl)piperdine and Maleimidopropionamyl-3,5-3′,5′tetraiodothyroninamide.

[0029] This invention is further directed to the an aerosol compositionfor delivery of a therapeutic agent to the pulmonary system of a hostcomprising an aerosolized aqueous solution containing a modifiedtherapeutic agent conjugated to a blood protein.

[0030] This invention is further directed to the use of a particulateformulation for delivery of a therapeutic agent to the pulmonary systemof a host comprising:

[0031] a dispersable dry powder containing a modified therapeutic agent,the modified therapeutic agent comprising a therapeutic agent and areactive group which reacts with amino groups, hydroxyl groups or thiolgroups on pulmonary components to form a stable covalent bond whereinsaid therapeutic agent is covalently bonded to a blood protein.

DETAILED DESCRIPTION OF THE INVENTION

[0032] Definitions

[0033] To ensure a complete understanding of the invention the followingdefinitions are provided:

[0034] Therapeutic Agents: Therapeutic agents are agents that have atherapeutic effect and inlcude peptides and non-peptide organicmolecules. Therapeutic agents include but are not limited to woundhealing agents, antibiotics, anti-infectives, anti-oxidants,chemotherapeutic agents, anti-cancer agents, anti-inflammatory agents,and antiproliferative drugs. Therapeutic agents also includeabortifacients, ace-inhibitor, α-adrenergic agonists, β-adrenergicagonists, α-adrenergic blockers, β-adrenergic blockers, adrenocorticalsteroids, adrenocortical supressants, adrenocorticotrophic hormones,alcohol deterrents, aldose reductase inhibitors, aldosteroneantagonists, 5-alpha reductase inhibitors, anabolics, analgesics,analgesics, analgesics, androgens, anesthetics, anesthetics, angiotensincoverting enzyme inhibitors, anorexics, antacids, anthelmintics,antiacne agents, antiallergic agents, antialopecia agents, antiamebicagents, antiandrogen agents, antianginal agents, antiarrhythmic agents,antiarterioscierotic agents, antiarthritic/antirheumatic agents,antiasthmatic agents, antibacterial agents, aminoglycosides,amphenicols, ansamycins, β-lactams, lincosamides, macrolides,polypeptides, tetracyclines, antibacterial agents,2,4-diaminopyrimidines, nitrofurans, quinolones and analogs,sulfonamides, sulfones, antibiotics, anticholelithogenic agents,anticholesteremic agents, anticholinergic agents, anticoagulant agents,anticonvulsant agents, antidepressant agents, hydrazides/hydrazines,pyrrolidones, tetracyclics, antidiabetic agents, biguanides, hormones,sulfonylurea derivatives, antidiarrheal agents, antiduretic agents,antidotes, antidote, antidote, antidote, antidote, antidyskinetic,antieczematic, antiemetic agents, antiepileptic agents, antiestrogenagents, antifibrotic agents, antiflatulent agents, antifungal agents,polyenes, allylamines, imidazoles, triazoles and antiglaucoma agents.

[0035] Other therapetic agents include anti-viral agents, anti-fusogenicagents, blood brain barrier peptides (BBB peptides), RGD peptides,glucagon-like peptides, antigonadotropin, antigout, antihemorrhagic andantihistaminic agents; alkylmaine derivatives, aminoalkyl ethers,ethylenediamine derivatives, piperazines and tricyclics,antihypercholesterolemic, antihyperlipidemic, anthyperlipidemic andantihyperlipoproteinemic agents, aryloxyalkanoic acid derivatives, bileacid sequesterants, hmg coa reductase inhibitors, nicotine acidderivatives, thyroid hormones/analogs, antihyperphosphatemic,antihypertensive agents, arlethanolamine derivatives,arloxypropanolamine derivatives, benzothiadiazine derivatives,n-carboxyalkyl derivatives, dihydropyridine derivatives, guanidinederivatives, hydrazines/phthalazines, imidazole derivatives, quaternaryammonium compounds, quinazolinyl piperazine derivatives, reserpinederivatives, sulfonamide derivatives, antihyperthyroid agents,antihypotensive agents, antihypothyroid agents, anti-infective agents,anti-inflammatory agents, anti-inflammatory agents, aminoarylcarboxylicacid derivatives, arylacetic acid derivatives, arylbutyric acidderivatives and arylcarboxylic acids.

[0036] Therapeutic agents also include arylpropionic acid derivatives,pyrazoles, pyrazolones, salicylic acid derivatives,thiazinecarboxamides, antileprotic, antileukemic, antilipemic,antilipidemic, antimalarial, antimanic, antimethemoglobinemic,antimigraine, antimycotic, antinauseant, antineoplastic and alkylatingagents, antimetabolites, enzymes, androgens, antiadrenals,antiandrogens, antiestrogens, lh-rh analogs, progestogens, adjunct,folic acid replenisher, uroprotective and antiosteporotic agents

[0037] Therapeutic agents also include antipagetic, antiparkinsonian,antiperistaltic, antipheochromocytoma, antipneumocystis, antiprostatichypertrophy, antiprotozoal, antiprozoal, antipruritic, antipsoriatic andantipsychotic agents, butyrophenes, phenothiazines, thioxanthenes,antipyretic, antirheumatic, antirickettsial, antiseborreheic andantiseptic/disinfectant agetns, alcohols, aldehydes, dyes, guanidines,halogens/halogen compounds, mercurial compounds, nitrofurans,peroxides/permanganates, phenols, quinolines, silver compounds, others,antispasmodic,antisyphilitic, antithrombotic, antitubercular, antitumor,antitussive, antiulcerative, antiurolithic, antivenin, antivertigo andantiviral agents, purines/pyrimidinomes, anxiolytic, arylpiperazines,benzodiazepine derivatives, carbamates, astringent, benzodiazepineantagonist, beta-blocker, bronchodilator, ephedrine derivatives, calciumchannel blockers, arylalkylamines, dihydropyridine derivatives,piperazine derivatives, calcium regulators, calcium supplements, cancerchemotherapy agents, capillary protectants, carbonic anhydraseinhibitors, cardiac depressants, cardiotonic, cathartic, cation-exchangeresin, cck antagonists, central stimulants,cerebral vasodilators,chelating agents, cholecystokinn antagonists, choleitholytic agents,choleretic agents, cholinergic agents, cholinesterase inhibitors,cholinesterase reactivators, cns stimulants, cognition activators,contraceptives, agents to control intraocular pressure,converting-enzyme inhibitors, coronary vasodilators, cytoprotectants,debriging agenta, decongestants, depigmentora, dermatitis herpretiformissuppresanta, diagnostic aids, digestive aids, diuretics,benthothiadiazine derivatives, organomercurials, pteridines, purines,steroids, sulfanamide derivatives, uracils, others, dopamine andreceptor agonists.

[0038] Therapeutic agents also include dopamine receptor antagonists,ectoparasiticides, electrolyte replenishers, emetics, enzymes, digestiveagents, mucolytic agents, penicillin inactivating agents, proteolyticagents, enzyme inducers, estrogen antagonists, expectorant gastric andpancreatic secreation stimulantd, gastric proton pump inhibitor, gastricsecretion inhibitord, glucocorticoidd, α-glucosidase inhibitord,gonad-stimulating principled, gonadotrophic hormoned, gout suppressant,growth hormone inhibitor, growth hormone releasing factor, growthstimulant, hematinic, hemolytic, demostatic, heparin antagonist,hepatoprotectant, histamine h₁-receptor antagonists, histamineh₂-receptor antagonists, hmg coa reductase inhibitor, hypnotic,hypocholesteremic and hypolipidemic agents.

[0039] Therapeutic agents also include hypotensive, immunomodulators,immunosuppressants, inotrophic agents, keratolytic agents, lactationstimulating hormone, laxative/cathargic, lh-rh agonists, lipotrophicagents, local anesthetics, lupus erythematosus suppressants, majortranquilizers, mineralocorticoids, minor tranquilizers, miotic agents,monoamine oxidase ihibitors, mucolytic agents, muscle relaxants,mydriatic agents, narcotic agents; analgesics, narcotic antagonists,nasal decongestants, neuroleptic agents, neuromuscular blocking agents,neuroprotective agents, nmda antagonists, nootropic agents, nsaidagents, opioid analgesics, oral contraceptives and ovarian hormones.

[0040] Therapeutic agents also include oxytocic agents, blood brainbarrier protiens, GP41 peptides, insulinotropic peptidesparasympathomimetic agents, pediculicides, pepsin inhibitors, peripheralvasodilators, peristaltic stimulants, pigmentation agents, plasma volumeexpanders, potassium channel activators/openers, pressor agents,progestogen, prolactin inhibitors, prostaglandin/prostaglandin analogs,protease inhibitors, proton pump inhibitors, 5α-reductase inhibitors,replenishers/supplements, respiratory stimulants, reverse transcriptaseinhibitors, scabicides, sclerosing agents, sedative/hypnotic agents,acyclic ureides, alcohols, amides, barbituric acid derivatives,benzodiazepine derivatives, bromides, carbamates, chloral derivatives,quinazolone derivatives and piperidinediones.

[0041] Therapeutic agents also include serotonin receptor agonists,serotonin receptor antagonists, serotonin uptake inhibitors, skeletalmuscle relaxants, somatostatin analogs, spasmolytic agents, stoolsofteners, succinylcholine synergists, sympathomimetics, thrombolytics,thyroid hormone, thyroid inhibitors, thyrotrophic hormone, tocolytic,topical protectants, uricosurics, vasodilators, vasopressors,vasoprotectants, vitamin/vitamin sources, antichitic, antiscorbutic andantixerophthalmic agents, enzyme co-factors, hematopoietic, prombogenicagents and xanthene oxidase inhibitors.

[0042] Diagnostic Imaging Agents: Diagnostic imaging agents are agentsuseful in imaging the mammalian vascular system and include such agentsas position emission tomography (PET) agents, computerized tomography(CT) agents, magnetic resonance imaging (MRI) agents, nuclear magneticimaging agents (NMI), fluroscopy agents and ultrasound contrast agents.Diagnostic agents of interest include radioisotopes of such elements asiodine (I), including ¹²³I, ¹²⁵I, ¹³¹I, etc., barium (Ba), gadolinium(Gd), technetium (Tc), including ⁹⁹Tc, phosphorus (P), including ³¹P,iron (Fe), manganese (Mn), thallium (TV), chromium (Cr), including ⁵¹Cr,carbon (C), including ¹⁴C, or the like, fluorescently labeled compounds,etc.

[0043] Wound Healing Agents: Wound healing agents are agents thatpromote wound healing. Wound healing agents include integrins, celladhesion molecules such as ICAM, ECAM, ELAM and the like, antibiotics,growth factors such as EGF, PDGF, IGF, bFGF, aFGF and KGF, fibrin,thrombin, RGD peptides and the like.

[0044] Antiproliferatives: Antiproliferatives include antimetabolites,topoisomerase inhibitors, folic acid antagonists like methotrexate,purine antagonists like mercaptopurine, azathioprine, and pyrimidineantagonists like fluorouracil, cytarabine and the like.

[0045] Antioxidants: Antioxidants are agents that prevents oxidativedamage to tissue and include aspartate, orotate, tacophenol derivative(vitamin E), and free radical scavengers such as SOD, glutathione andthe like.

[0046] Mammalian cells are continuously exposed to activated oxygenspecies such as superoxide, hydrogen peroxide, hydroxyl radical, andsinglet oxygen. These reactive oxygen intermediates are generated invivo by cells in response to aerobic metabolism, catabolism of drugs andother xenobiotics, ultraviolet and x-ray radiation, and the respiratoryburst of phagocytic cells (such as white blood cells) to kill invadingbacteria such as those introduced through wounds. Hydrogen peroxide, forexample, is produced during respiration of most living organismsespecially by stressed and injured cells.

[0047] Active oxygen species can injure cells. An important example ofsuch damage is lipid peroxidation which involves the oxidativedegradation of unsaturated lipids. Lipid peroxidation is highlydetrimental to membrane structure and function and can cause numerouscytopathological effects. Cells defend against lipid peroxidation byproducing radical scavengers such as superoxide dismutase, catalase, andperoxidase. Injured cells have a decreased ability to produce radicalscavengers. Excess hydrogen peroxide can react with DNA to causebackbone breakage, produce mutations, and alter and liberate bases.Hydrogen peroxide can also react with pyrimidines to open the 5,6-doublebond, which reaction inhibits the ability of pyrimidines to hydrogenbond to complementary bases, Hallaender et al. (1971). Such oxidativebiochemical injury can result in the loss of cellular membraneintegrity, reduced enzyme activity, changes in transport kinetics,changes in membrane lipid content, and leakage of potassium ions, aminoacids, and other cellular material.

[0048] Antioxidants have been shown to inhibit damage associated withactive oxygen species. For example, pyruvate and other alpha-ketoacidshave been reported to react rapidly and stoichiometrically with hydrogenperoxide to protect cells from cytolytic effects, O'Donnell-Tormey etal., J. Exp. Med., 165, pp. 500-514 (1987).

[0049] Anti-infective Agents: Anti-infective agents are agents thatinhibit infection and include anti-viral agents, anti-fungal agents andantibiotics.

[0050] Anti-Viral Agents: Anti-viral agents are agents that inhibitvirus and include vidarabine, acyclovir and trifluorothymidine.

[0051] Anti-Fungal Agents: Anti-fungal agents are agents that inhibitfungal growth. Anti-fungal agents include anphoterecin B, myconazole,terconazole, econazole, isoconazole, thioconazole, biphonazole,clotrimazole, ketoconazole, butaconazole, itraconazole, oxiconazole,phenticonazole, nystatin, naphthyphene, zinoconazole, cyclopyroxolamineand fluconazole.

[0052] Antibiotics: Antibiotics are natural chemical substances ofrelatively low molecular weight produced by various species ofmicroorganisms, such as bacteria (including Bacillus species),actinomycetes (including Streptomyces) and fungi, that inhibit growth ofor destroy other microorganisms. Substances of similar structure andmode of action may be synthesized chemically, or natural compounds maybe modified to produce semi-synthetic antibiotics. These biosyntheticand semi-synthetic derivatives are also effective as antibiotics. Themajor classes of antibiotics are (1) the beta-lactams, including thepenicillins, cephalosporins and monobactams; (2) the aminoglycosides,e.g. gentamicin, tobramycin, netilmycin, and amikacin; (3) thetetracyclines; (4) the sulfonamides and trimethoprim; (5) thefluoroquinolones, e.g. ciprofloxacin, norfloxacin, and ofloxacin; (6)vancomycin; (7) the macrolides, which include for example, erythromycin,azithromycin, and clarithromycin; and (8) other antibiotics, e.g., thepolymyxins, chloramphenicol and the lincosamides.

[0053] Antibiotics accomplish their anti-bacterial effect throughseveral mechanisms of action which can be generally grouped as follows:(1) agents acting on the bacterial cell wall such as bacitracin, thecephalosporins, cycloserine, fosfomycin, the penicillins, ristocetin,and vancomycin; (2) agents affecting the cell membrane or exerting adetergent effect, such as colistin, novobiocin and polymyxins; (3)agents affecting cellular mechanisms of replication, informationtransfer, and protein synthesis by their effects on ribosomes, e.g., theaminoglycosides, the tetracyclines, chloramphenicol, clindamycin,cycloheximide, fucidin, lincomycin, puromycin, rifampicin, otherstreptomycins, and the macrolide antibiotics such as erythromycin andoleandomycin; (4) agents affecting nucleic acid metabolism, e.g., thefluoroquinolones, actinomycin, ethambutol, 5-fluorocytosine,griseofulvin, rifamycins; and (5) drugs affecting intermediarymetabolism, such as the sulfonamides, trimethoprim, and thetuberculostatic agents isoniazid and para-aminosalicylic acid. Someagents may have more than one primary mechanism of action, especially athigh concentrations. In addition, secondary changes in the structure ormetabolism of the bacterial cell often occur after the primary effect ofthe antimicrobial drug.

[0054] Anti-Cancer Agents: Anti-cancer agents (chemotherapeutic agents)are natural or synthetic molecules which are effective against one ormore forms of cancer. This definition includes molecules which by theirmechanism of action are cytotoxic (anti-cancer chemotherapeutic agents),those which stimulate the immune system (immune stimulators) andmodulators of angiogenesis. The outcome in either case is the slowing ofthe growth of cancer cells.

[0055] Anti-cancer therapy include radioactive isotopes such as ³²P usedin the treatment of polycythemia vera and in chronic leukemia.Radioactive phosphorus has a biological half-life of about 8 days inhumans. It emits beta rays that exert a destructive effect on therapidly multiplying cells. ³²P is usually administered in doses of about1 mc daily for 5 days. Either the oral or intravenous route may be usedand the doses are not greatly different. Radioactive iodine ¹³¹I,radioactive gold ¹⁹⁸Au, and other isotopes are not as useful as ³²P.Nevertheless, ¹³¹I has some limited applications in metastatic thyroidcarcinoma. Other radioactive isotopes can be used with our technologyeither as complexes of radioactive metal such as ⁵¹Cr, ⁵²Mn, ⁵²Mg, ⁵⁷Ni,⁵⁵Co and ⁵⁶P, ⁵⁵Fe, ¹⁰³Pd, ¹⁹²Ir, ⁸⁴Cu and ⁶⁷Cu or as chelates of thesemetals using bifunctional chelating agents like (BFCs),6-[p-(bromoacetamido)benzyl]-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraaceticacid (BAT),6-[p-(isothiocyanato)benzyl]-1,4,8,11-tetraazacyclotetradecane-1,4,8,11-tetraaceticacid (SCN-TETA), 4-[(1,4,8,11-tetraazacyclotetradec-1-yl)methyl]benzoicacid (CPTA), and1-[(1,4,7,10,13-pentaazacyclopentadec-1-yl)methyl]benzoic acid (PCBA).

[0056] Numerous drugs fall into the category of chemotherapeutic agentsuseful in the treatment of neoplastic disease that are amenable to theembodiment of this application. Such agents derivitized with thistechnology can include anti-metabolites such as metotrexate (folic acidderivatives), fluoroaucil, cytarabine, mercaptopurine, thioguanine,petostatin (pyrimidine and purine analogs or inhibitors), a variety ofnatural products such as vincristine and vinblastine (vinca alkaloid),etoposide and teniposide, various antibiotics such as miotomycin,plicamycin, bleomycin, doxorubicin, danorubicin, dactomycin; a varietyof biological response modifiers including interferon-alpha; a varietyof miscellaneous agents and hormonal modulators including cisplatin,hydroxyurea, mitoxantorne, procarbozine, aminogultethimide, prednisone,progestins, estrogens, antiestorgens such as tamoxifen, androgenicsteroids, antiadrogenic agents such as flutamide, gonadotropin releasinghormones analogs such as leuprolide, the matrix metalloproteaseinhibitors (MMPIs) as well as anti-cancer agents including Taxol(paclitaxel) and related molecules collectively termed taxoids, taxinesor taxanes.

[0057] Included within the definition of “taxoids” are variousmodifications and attachments to the basic ring structure (taxoidnucleus) as may be shown to be efficacious for reducing cancer cellgrowth and which can be constructed by organic chemical techniques knownto those skilled in the art.

[0058] Chemotherapeutics include podophyllotoxins and their derivativesand analogues. Another important class of chemotherapeutics useful inthis invention are camptothecins.

[0059] Another preferred class of chemotherapeutics useful in thisinvention are the anthracyclines (adriamycin and daunorubicin).

[0060] Another important class of chemotherapeutics are compounds whichare drawn from the following list: Taxotere, Amonafide, Illudin S,6-hydroxymethylacylfulvene Bryostatin 1,26-succinylbryostatin 1,Palmitoyl Rhizoxin, DUP 941, Mitomycin B, Mitomycin C, Penclomedine,angiogenesis inhibitor compounds, Cisplatin hydrophobic complexes suchas 2-hydrazino-4,5-dihydro-1H-imidazole with platinum chloride and5-hydrazino-3,4-dihydro-2H-pyrrole with platinum chloride, vitamin A,vitamin E and its derivatives, particularly tocopherol succinate.

[0061] Other compounds useful in the invention include:1,3-bis(2-chloroethyl)-1-nitrosurea (“carmustine” or “BCNU”),5-fluorouracil, doxorubicin (“adriamycin”), epirubicin, aclarubicin,Bisantrene(bis(2-imidazolen-2-ylhydrazone)-9,10-anthracenedicarboxaldehyde,mitoxantrone, methotrexate, edatrexate, muramyl tripeptide, muramyldipeptide, lipopolysaccharides, vidarabine and its 2-fluoro derivative,resveratrol, retinoic acid and retinol, carotenoids, and tamoxifen.

[0062] Other chemotherapeutic agents useful in the application of thisinvention include: Decarbazine, Lonidamine, Piroxantrone,Anthrapyrazoles, Etoposide, Camptothecin, 9-aminocamptothecin,9-nitrocamptothecin, camptothecin-11 (“Irinotecan”), Topotecan,Bleomycin, the Vinca alkaloids and their analogs [Vincristine,Vinorelbine, Vindesine, Vintripol, Vinxaltine, Ancitabine],6-aminochrysene, and Navelbine.

[0063] Other compounds useful in the application of the invention aremimetics of taxol, eleutherobins, sarcodictyins, discodermolides andepothiolones.

[0064] Antineoplastic Agents—Antineoplastic agents are anti-canceragents such as fluoropyrimidines, pyrimidine nucleosides, purines,platinum analogs, anthracyclines/anthracenediones, podophyllotoxins,camptothecins, hormones and hormonal analogs, enzymes, proteins andantibodies, vinca alkaloids, taxanes, atihormonal agents, antifolates,antimicrotubule agents, alkylating agents (classical and non-classical),antimetabolites, antibiotics, topoisomerase inhibitors, antivirals, andmiscellaneous cytotoxic agents, for example hydroxyurea, mitotane,fusion toxins, PZA, bryostatin, retinoids, butyric acid and derivatives,pentosan, fumagillin, and others. The objective of all antineoplasticdrugs is to eliminate (cure) or to retard the growth and spread(remission) of cancer cells. The majority of the above listedantineoplastic agents pursue this objective by possessing primarycytotoxic activity, effecting a direct kill on the cancer cells. Otherantineoplastic drugs stimulate the body's natural immunity to effectcancer cell death.

[0065] Matrix metalloprotease inhibitors (MMPIs)—Also known as matrixmetalloproteinase inhibitors, MMPIs are inhibitors of the matrixmetalloproteases. The metalloproteases are a family of enzymescontaining zinc at the active site, which facilitate the catalytichydrolysis of various protein substrates. A subfamily of themetalloprotease family is known as the matrix metalloproteases (MMPs)because these enzymes are capable of degrading the major components ofarticular cartilage and basement membranes. The matrix metalloproteasesinclude stromelysin, collagenase, matrylisin and gelatinase, amongother. The action of matrix metalloptoreases is inhibited by MMPIs usedin the preparation of the derivatized MMPIs of the present invention.Some characterized MMPs and their preferred substrates are illustratedin the following table.

[0066] The nomenclature used to describe the interaction of proteasesand their substrates is widely used in the protease literature. In thissystem, the binding site for a polypeptide substrate on a protease isenvisioned as a series of subsites; each subsite interacts with oneamino acid reside of the substrate. By convention, the substrate aminoacid residues are called P (for peptide); the subsites on the proteasethat interact with the substrate are called S (for subsite). Thesubsites are in the catalytic or active site of the protease. The aminoacid residues on the amino-terminal side of the scissile bond (bond thatis cleaved on the substrate) are numbered P₁, P₂, P₃, etc., and theresidues on the carboxy-terminal side of the scissile bond are numberedP₁′, P₂′, P₃′, etc. The residues can be numbered up to P₆ on each sideof the scissile bond. The subsites on the protease are termed S₃, S₂,S₁, S₁′, S₂′, S₃′, etc. to complement the substrate residues thatinteract with the enzyme.

[0067] Characterized MMPs and their preferred substrates. MATRIX MMPPREFERRED METALLOPROTEINASE NUMBER SUBSTRATE CLASS I Interstitialcollagenase 1 Fibrillar collagens, type I, II, III Neutrophil (PMN) 8Fibrillar collagens, type I, II, III collagenase Collagenase-3 13Fibrillar collagens, type I, II, III Collagenase-4 18 CLASS IIGelatinase A (72 kDa) 2 Collagen types IV, V, gelatin Gelatinase B (92kDa) 9 Collagen types IV, V, gelatin Metalloelastase 12 Elastin CLASSIII Stromelysin-1 3 Laminin, fibronectin, proteoglycans Stromelysin-2 10Laminin, fibronectin, proteoglycans Matrylisin (pump) 7 Laminin,fibronectin, proteoglycans NON-CLASSIFIED Stromelysin-3 11 1-antitrypsinMembrane-type MMP 14-17 Pro-gelatinase A

[0068] In this application, the term MMPI should be understood toinclude matrix metalloprotease inhibitors as well as analogs thereof. Inaddition, the term MMPI includes optical isomers and diastereomers; aswell as the racemic and resolved, enantiomerically pure R and Sstereoisomers; as well as other mixtures of the R and S stereoisomersand pharmaceutically acceptable salts thereof.

[0069] Oxytocin—Oxytocin is a hormone involved in the enhancement oflactation, contraction of the uterus, and relaxation of the pelvis priorto childbirth. Oxytocin secretion in nursing women is stimulated bydirect neural feedback obtained by stimulation of the nipple duringsuckling. Its physiological effects include the contraction of mammarygland myoepithelial cells, which induces the ejection of milk frommammary glands, and the stimulation of uterine smooth muscle contractionleading to childbirth. Oxytocin causes myoepithelial cells surroundingsecretory acini of mammary glands to contract, pushing milk throughducts. In addition, it stimulates the release of prolactin, andprolactin is trophic on the breast and stimulates acinar formation ofmilk.

[0070] Cholecystokinin (CCK)—CCK is a polypeptide of 33 amino acidsoriginally isolated from pig small intestine that stimulates gallbladdercontraction and bile flow and increases secretion of digestive enzymesfrom pancreas. It exists in multiple forms, including CCK4 and CCK-8,with the octapeptide representing the dominant molecular species showingthe greatest activity. It belongs to the CCK/gastrin peptide family andis distributed centrally in the nervous system and peripherally in thegastrointestinal system. It has many biological roles, includingstimulation of pancreatic secretion, gall bladder contraction andintestinal mobility in the GI tract as well as the possible mediation ofsatiety and painful stimuli.

[0071] Antihypertensive Agents—Antihypertensive agents are variousagents that can be used to treat hypertension, including but not limitedto enalapril, acebutolol, and doxazosin. Enarlapril is a pro-drug thatis activated to the angiotensin-converting enzyme (ACE) inhibitor,enalaprilat. This pro-drug inhibits the conversion of angiotensin I toangiotensin II and exerts an antihypertensive effect by suppressing therenin-angiotensin-aldosterone system. Acebutolol is in a class of drugscalled beta-blockers, which affect the heart and circulatory system.Acebutolol is used to lower blood pressure, lower heart rate, and reduceangina (chest pain). Doxazosin is a member of the alpha blocker familyof drugs used to lower blood pressure in people with hypertension.Doxazosin is also used to treat symptoms of benign prostatic hyperplasia(BPH). Doxazosin works by relaxing blood vessels so that blood passesthrough them more easily, which helps to lower blood pressure.

[0072] Methylprednisolone—Methylprednisolone is a synthetic steroid thatsuppresses acute and chronic inflammation. In addition, it stimulatesgluconeogenesis, increases catabolism of proteins and mobilization offree fatty acids. In addition, it potentiates vascular smooth musclerelaxation by beta adrenergic agonists, and may alter airwayhyperactivity. It is also a potent inhibitor of the inflammatoryresponse.

[0073] GP-41 Peptides—GP-41 is an HIV transmembrane protein which hasbeen shown to be essential for the virus to fuse with and infect healthycells.

[0074] Anti-viral and antifusogenic peptides: Anti-viral peptides refersto peptides that inhibit viral infection of cells, by, for example,inhibiting cell-cell fusion or free virus infection. The route ofinfection may involve membrane fusion, as occurs in the case ofenveloped viruses, or some other fusion event involving viral andcellular structures. Peptides that inhibit viral infection by aparticular virus may be referenced with respect to that particularvirus, e.g., anti-HIV peptide, anti-RSV peptide, etc. Antifusogenicpeptides are peptides demonstrating an ability to inhibit or reduce thelevel of membrane fusion events between two or more entities, e.g.,virus-cell or cell-cell, relative to the level of membrane fusion thatoccurs in the absence of the peptide.

[0075] In particular, anti-viral and antifusogenic peptides include theDP107 and DP178 peptides and analogs thereof, as well as peptidescomprised of amino acid sequences from other (non-HIV) viruses thatcorrespond to the gp41 region of HIV from which DP107 and DP178 arederived, and that exhibit anti-viral or anti-fusogenic activity. Thhesepeptides can exhibit anti-viral activity against not only HIV, but otherviruses including human respiratory syncytial virus (RSV), humanparainfluenza virus (HPV), measles virus (MeV) and simianimmunodeficiency virus (SIV).

[0076] In particular, anti-HIV peptides refer to peptides that exhibitanti-viral activity against HIV, including inhibiting CD-4+ cellinfection by free virus and/or inhibiting HIV-induced syncytia formationbetween infected and uninfected CD-4+ cells. Anti-SIV peptides arepeptides that exhibit anti-viral activity against SIV, includinginhibiting of infection of cells by the SIV virus and inhibitingsyncytia formation between infected and uninfected cells. Anti-RSVpeptides are peptides that exhibit anti-viral activity against RSV,including inhibiting mucous membrane cell infection by free RSV virusand syncytia formation between infection and uninfected cells. Anti-HPVpeptides are peptides that exhibit anti-viral activity against HPV,including inhibiting infection by free HPV virus and syncytia formationbetween infected and uninfected cells. Anti-MeV peptides are peptidesthat exhibit anti-viral activity against MeV, including inhibitinginfection by free MeV virus and syncytia formation between infected anduninfected cells.

[0077] Blood Brain Barrier (BBB) Peptides—The “blood-brain barrier” is alayer of cells that controls which substances may penetrate from thegeneral circulation into the brain. BBB proteins can traverse thisbarrier through protein transduction. Small sections of these proteins(10-16 residues long), i.e. BBB peptides, are responsible for thistransduction.

[0078] RGD Peptides: The RGD peptide for conjugation to tissues or fixedendogenous proteins in accordance with the present invention includes asequence of amino acids, preferably naturally occurring L-amino acidsand glycine, having the following formula:

R₁-Arg-Gly-Asp-R₂

[0079] In this formula, R₁ and R₂ represent an amino acid or a sequenceof more than one amino acid or a derivatized or chemically modifiedamino acid or more than one derivatized or chemically modified aminoacids.

[0080] Insulinotropic Peptides: Insulinotropic peptides (ITPs) arepeptides with insulinotropic activity. Insulinotropic peptidesstimulate, or cause the stimulation of, the synthesis or expression ofthe hormone insulin. Such peptides include precursors, analogues,fragments of peptides such as Glucagon-like peptide, exendin 3 andexendin 4 and other peptides with insulinotropic activity.

[0081] Glucagon-Like Peptide: Glucagon-Like Peptide (GLP) and GLPderivatives are intestinal hormones which generally simulate insulinsecretion during hyperglycemia, suppresses glucagon secretion,stimulates (pro) insulin biosynthesis and decelerates gastric emptyingand acid secretion. Some GLPs and GLP derivatives promote glucose uptakeby cells but do not simulate insulin expression as disclosed in U.S.Pat. No. 5,574,008 which is hereby incorporated by reference.

[0082] Pulmonary Condition: A pulmonary condition is a disease whichaffects lung function. Such conditions may result from a defect in agene or genes associated with lung function (e.g., cystic fibrosis),asthma, allergies, an immune or autoimmune disorder, a microbialinfection (e.g. bacterial, viral, fungal or parasitic infection), or amechanical injury to the lungs.

[0083] Exemplary pulmonary conditions contemplated by the subjectinvention include cystic fibrosis, asthmatic bronchitis, tuberculosis,bronchitis, bronchiectasis, laryngotracheobronchitis, bronchiolitis,emphysema, bronchial pneumonia, allergic bronchopneumonia, viralpneumonia, pertussis, diphtheria, spasmodic croup, pulmonary phthisis,encephalitis with retained secretions, and pulmonary edema. Otherpulmonary conditions, such as those which develop as a result of injuryor surgery (e.g., after tracheotomy), as well as those associated withinsufficient surfactant secretion in the lungs of premature infants, arealso contemplated by the subject invention. Pulmonary conditionsamenable to treatment by the subject method may also develop as a resultof activity associated with inhalation of particulate matter e.g.smoking, exposure to construction areas or other high dust areas,occupational hazards associated with inhalation of particulates,exposure to environmental particulates (e.g. smog, pollen, asbestos,siliconis), pulmonary delivery of pharmaceutical agents (e.g.bronchodilators) or inhalation of cocaine.

[0084] Other pulmonary conditions include diffuse parenchymal lungdisease from infectious cases, such as cytomegaloviral pneumonia ormiliary tuberculosis, drug-induced lung disease (after administration ofpenicillin, nitrofurantoin), neoplastic lung disease having lymphangiticspread pattern or bronchoalveolar cell carcinoma, granulomatous disease(infectious or noninfectious), hypersensitivity pneumonitis,histoplasmosis, tuberculosis, idiophatic pulmonary fibrosis (akacryptogenic fibrosing alveolitis), hereditary pulmonary disorders, suchas alveolar microlithiasis and bronchiectasis, eosinophilic granuloma,lympphangioleimyomatosis, and plumonary alveolar proteinosis disorder.

[0085] Symptoms of a Pulmonary Condition: Symptoms of a pulmonarycondition are symptoms associated with any of the pulmonary conditionsdescribed above. The classic symptoms associated with such pulmonaryconditions may include coughing, exertional dyspnea, wheezing, chestpain and purulent sputum production. Other components of the syndromewhich may accompany a pulmonary condition include hypoxia, CO₂ narcosis,hyperventilation, decreased expiration volume, and decreased lungcapacity.

[0086] Pulmonary Fluid: Pulmonary fluid is the fluid which bathes theapical surface of the lung epithelium, particularly the alveolarepithelium and contains fixed and mobile pulmonary fluid components.

[0087] Pulmonary Delivery Agent: Pulmonary delivery agents are agentsthat may be delivered to the lungs. Such agents include therapeuticagents.

[0088] Fixed Pulmonary Components: Fixed pulmonary components arenon-mobile pulmonary components and include tissues, membrane receptors,interstitial proteins, fibrin proteins, collagens, platelets,endothelial cells, epithelial cells and their associated membrane andmembraneous receptors, somatic body cells, skeletal and smooth musclecells, neuronal components, osteocytes and osteoclasts.

[0089] Mobile Pulmonary Components: Mobile pulmonary components arepulmonary components that do not have a fixed situs for any extendedperiod of time, generally not exceeding 5, more usually one minute.Mobile pulmonary components are components of the pulmonary or lungfluid and include such soluble proteins such as immunoglobulins, serumalbumin, ferritin, transferrin and the like.

[0090] Blood Components: Blood components may be either fixed or mobile.Fixed blood components are non-mobile blood components and includetissues, membrane receptors, interstitial proteins, fibrin proteins,collagens, platelets, endothelial cells, epithelial cells and theirassociated membrane and membraneous receptors, somatic body cells,skeletal and smooth muscle cells, neuronal components, osteocytes andosteoclasts and all body tissues especially those associated with thecirculatory and lymphatic systems. Mobile blood components are bloodcomponents that do not have a fixed situs for any extended period oftime, generally not exceeding 5, more usually one minute. These bloodcomponents are not membrane-associated and are present in the blood forextended periods of time and are present in a minimum concentration ofat least 0.1 μg/ml. Mobile blood components include serum albumin,transferrin, ferritin and immunoglobulins such as IgM and IgG. Thehalf-life of mobile blood components is at least about 12 hours.

[0091] Inhaler Device: An inhaler device is any device useful in theadministration of the inhalable medicament of the invention. Examples ofinhaler devices include nebulizers, metered dose inhalers, dry powderinhalers, intermittent positive pressure breathing apparatuses,humidifiers, bubble environments, oxygen chambers, oxygen masks andartificial respirators.

[0092] Reactive Groups: Reactive groups are chemical groups capable offorming a covalent bond. Such reactive groups are coupled or bonded to atherapeutic or diagnositic agent. Reactive groups will generally bestable in an aqueous environment and will usually be carboxy,phosphoryl, or convenient acyl group, either as an ester or a mixedanhydride, an imidate or maleimide, thereby capable of forming acovalent bond with functionalities such as an amino group, a hydroxy ora thiol at the target site on pulmonary components. For the most part,the esters will involve phenolic compounds, or be thiol esters, alkylesters, phosphate esters, or the like. Prefereably, the reactive groupwill be a maleimide group.

[0093] Functionalities: Functionalities are groups on pulmonarycomponents to which reactive groups on modified therapeutic agents reactto form covalent bonds. Functionalities include hydroxyl groups forbonding to ester reactive entities; thiol groups for bonding tomaleimides, imidates and thioester groups; amino groups for bonding tocarboxy, phosphoryl or acyl groups and carboxyl groups for bonding toamino groups.

[0094] IC₅₀: Concentration of an enzyme inhibitor at which 50% of theenzymatic activity is inhibited.

[0095] Protective Groups: Protective groups are chemical moietiesutilized to protect reactive entities from reacting with otherfunctionalities. Various protective groups are disclosed in U.S. Pat.No. 5,493,007 which is hereby incorporated by reference. Such protectivegroups include acetyl, fluorenylmethyloxycarbonyl (FMOC), t-butyloxycarbonyl (BOC), benzyloxycarbonyl (CBZ), and the like. For small organicmolecules all protecting groups like tetrahydropyranyl (THP), all silylderivatives, acetals, thioacetals and the like.

[0096] Linking Groups: Linking groups are chemical moieties that link orconnect reactive groups to therapeutic agents. Linking groups maycomprise one or more alkyl moeities, alkoxy moeity, alkenyl moeity,alkynyl moeity or amino moeity substituted by alkyl moeities, cycloalkylmoeity, polycyclic moeity, aryl moeity, polyaryl moeities, substitutedaryl moeities, heterocyclic moeities, and substituted heterocyclicmoeities. Linking groups may also comprise poly ethoxy amino acids, suchas AEA ((2-amino) ethoxy acetic acid) or a preferred linking group AEEA([2-(2-amino) ethoxy)] ethoxy acetic acid.

[0097] Sensitive functional groups—A sensitive functional group is agroup of atoms that represents a potential reaction site on atherapeutic agent. If present, a sensitive functional group may bechosen as the attachment point for the linking group-reactive groupmodification. Sensitive functional groups include but are not limited tocarboxyl, amino, thiol, and hydroxyl groups.

[0098] Modified Therapeutic and Diagnostic Agents—Modified therapeuticand diagnostic agents are agents that have been modified by attaching areactive group. The reactive group may be attached to the therapeuticagent either via a linking group, or optionally without using a linkinggroup. Modified therapeutic and diagnostic agents may be administered invivo such that conjugation with blood or pulmonary components occurs invivo, or they may be first conjugated to blood or pulmonary componentsin vitro and the resulting conjugated therapeutic agent (as definedbelow) administered in vivo.

[0099] Conjugated Therapeutic and Diagnostic Agents—Conjugatedtherapeutic and diagnostic agents are modified therapeutic anddiagnostic agents that have been conjugated to a blood or pulmonarycomponent via a covalent bond formed between the reactive group of themodified therapeutic agent and the functionalities of the pulmonarycomponent, with or without a linking group. As used throughout thisapplication, the term “conjugated therapeutic agent” can be made morespecific to refer to particular conjugated therapeutic agents, forexample “conjugated antihistamine.”

[0100] Taking into account these definitions, the present invention isdirected to modified therapeutic and diagnostic agents capable ofreacting with available functionalities on pulmonary or blood componentsvia covalent linkages. The invention is also directed to methods ofmaking the modified agents and their use. The modified therapeuticagents of the present invention are capable of reacting in vivo to formconjugates with pulmonary and/or blood components, such as pulmonary orblood proteins, thereby extending the half-life and improvingbioavailability of the therapeutic agent without deterioiusly alteringthe agent's therapeutic effect. In preferred embodiments of thisinvention, the functionality on the protein will be a thiol group andthe reactive group on the modified therapeutic agent will be amaleimido-containing group such as gamma-maleimide-butyralamide (GMBA),maleimidopropionic acid (MPA) or maleimide-benzoyl-succinimide (MBS).

[0101] The invention in one aspect contemplates delivery of the modifiedagents to the blood of a host for conjugation to blood components,including blood proteins. While pulmonary administration is furtherdescribed as such a route of administration, it will be understood thatthe invention is not limited to such routes of adminstration, and alsocontemplated administration of the modified agents to a patient's bloodstream using other methods, including parenterally, such asintravenously (IV), intraarterially (IA), intramuscularly (IM),subcutaneously (SC) and the like.

[0102] For pulmonary delivery, a wide variety of devices and carriermolecules have been utilized to enhance pulmonary drug delivery and canbe used with the modified agents of the present invention. These devicesand methods include metered dosing, carriers such as liposomes (Meisneret al, 1989) actide/glycolide copolymer (PLGA) nanospheres (Niwa et al.,1995), albumin microspheres (Feinstein et al., 1990), and other physicalart forms to created aerosols or nanoparticulates.

[0103] A new type of inhalation aerosol, characterized by particles ofsmall mass density and large size, has permitted the highly efficientdelivery of inhaled therapeutics (e.g. insulin, testosterone) into thesystemic circulation. Particles with mass densities less than 0.4 gramper cubic centimeter and mean diameters exceeding 5 micrometers havebeen reported to avoid the lungs' natural clearance mechanisms providinghigher bioavailability than that of conventional inhaled therapeuticparticles. (Edwards et al., 1997). For most of these therapies, aerosolsare designed to comprise small spherical droplets or particles of massdensity near 1 g/cm³ and mean geometric diameter between approximately 1and 3 micron, suitable for particle penetration into the airways or lungperiphery.

[0104] Studies performed primarily with liquid aerosols have shown thatthese characteristics of inhaled aerosols lead to optimal therapeuticeffect, both for local and systemic therapeutic delivery. Inefficientdrug delivery can still arise, owing to excessive particle aggregationin an inhaler, deposition in the mouth and throat, and overly rapidparticle removal from the lungs by mucocilliary or phagocytic clearancemechanisms.

[0105] To address these problems, particle surface chemistry and surfaceroughness are traditionally manipulated. Recent data indicate that majorimprovements in aerosol particle performance may also be achieved bylowering particle mass density and increasing particle size, sincelarge, porous particles display less tendency to agglomerate than(conventional) small and nonporous particles. Also, large, porousparticles inhaled into the lungs can potentially release therapeuticsubstances for long periods of time by escaping phagocytic clearancefrom the lung periphery, thus enabling therapeutic action for periodsranging from hours to many days. (Edwards et al., 1998)

[0106] It has been previously reported that specific transport receptorsfor albumin (GP60, albondin) exist in the endothelium that function asunique albumin carriers (U.S. Pat. No. 5,254,342). These transcytosisproteins facilitate the movement of albumin and albumin carriers acrossthe lining of the airway and result in extensive plasma levels of theseproteins or protein carriers. As further described, modified agentsaccording to the present invention can be prepared that react withalbumin, and upon pulmonary delivery, the resulting conjugates can passto the bloodstream via such carriers.

[0107] Pulmonary drug delivery is also advantageous for local treatmentof the lung in that it promotes an increase in drug retention-time inthe lung and more importantly, a reduction in extrapulmonaryside-effects, invariably resulting in enhanced therapeutic efficacies.(Shek, 1994). A key advantage of pulmonary delivery includes reducedsystemic toxicity and increased drug concentration at the site of action(e.g. infection or inflammation site. (Stout and Derendorf, 1987).

[0108] The use of in vivo or ex vivo bioconjugation associated withpulmonary drug delivery includes the following non-limited benefits.Retention of the drug at the site of placement is enhanced due tocovalently attachment of the drug to the airway site. Additionally,prolonged duration of action of the drug is made possible, both in thelung by in situ attachment to soluble proteins for localizedintrapulmonary activity, as well as systemic absorption and conjugationto blood proteins.

[0109] Drug stability is improved, both locally and systemically, asconjugation affords protection against enzymatic degradation that occursin the pulmonary mucosal fluid or in the plasma. Also, in the deeplungs, alveolar macrophages can rapidly deposited particles; thereactivity of the modified agents of the invention with epithelial cellswill allow for localized retention of the agent.

[0110] Localized delivery to the pulmonary tissues also reduces toxicityand reduces systemic exposure as there is no first-pass liver effect.Systemic delivery also exhibits reduced extravascular side effectsthrough conjugation to, for example, albumin, due to, for example, thelimitation of hepatic, central nervous system (CNS) or renal toxicitydue to the limited clearance of albumin into these organs.

[0111] Pulmonary delivery of the modified agents of the invention alsoprovides advantages of improved patient compliance due to prolongedduration of action of the modified agents. In turn, cost benefits can beachieved through, e.g., reduced costs of goods per course of therapy dueto prolonged duration of action, and outpatient use of medications thatwould otherwise have limited use or complicated dosing titrationschedules. Pulmonary delivery can also reduce difficulties associatedwith oral dosing, including low solubility, interactions with food, andlow bioavailability.

[0112] A further advantage of pulmonary delivery of modified agents ofthe present invention is the ability to deliver systemically-largemacromolecule drugs, such as insulin, growth hormones, beta-interferon,calcitonin, and others that, due to their large size and instability,are typically delivered by injection. The present invention provides analternative and more convenient route of adminstering these drugs. Inaddition, many drugs and therapeutic peptides are more stable in solid,dry form, rather than solubilized. Dry pulmonary formulations of suchdrugs modified according to the present invention provide for a morestable form of the drug, as well as the convenience of pulmonaryadministration.

[0113] 1. Therapeutic Agents

[0114] A wide variety of therapeutic agents are contemplated for use inthe present invention, including peptide therapeutics and small organicmolecules, provided they can be modified as described.

[0115] In addition to therapeutic agents discussed above, the followingtherapeutic agents are within the scope of this invention.

[0116] Sympathomimetic compounds mimic the action of endogenouscatecholamines (adrenalinelike neurotransmitters) at peripheralsympathetic neurons in addition to CNS effects. Adrenaline systemscontrol important body functions like blood pressure regulation andwakefulness. A vast panoply of compounds has been developed that allowsone to selectively tweak these systems. These include agonists, used asdecongestants and antiasthmatics and antagonists, used asantihypertensives).

[0117] Selective agents include alpha agonists such as phenylephrine(Neo-Synephrine) and more lipophilic agents such as naphazoline.Clonidine, an antihypertensive, is sometimes used in ethanol withdrawal,and has been tried in cigarette smoking cessation. It probably has a netantagonist effect through autoreceptors, i.e. presynaptic receptorsdetect an excess of adrenergic agonist and decrease norepinephrinerelease. Ibopamine, a cardiotonic, has diuretic and dopamine agonistactivity.

[0118] The beta agonists are popular antiasthma medications.Isoproterenol and dobutamine are fairly selective for the beta1receptor; the latter is used as a cardiotonic.

[0119] Alpha blockers such as phentolamine and prazosin are also used asantihypertensives. Yohimbine, a selective alpha2 blocker, is a popularaphrodisiac for males, purported to prolong or intensify erection.Alpha2 receptors are inhibitory, so that inhibiting them produces astimulating effect. Alpha2 and alpha3 receptors are alsopresent in fatstorage sites; antagonizing them may provide a way to promote fatcatabolism without resorting to central stimulants.

[0120] Beta antagonists include propranolol, which has been used forsome time to calm the nerves in event-specific anxiety (stage fright) aswell as its more traditional role in hypertension. Acebutolol is acardioselective beta blocker used in angina.

Antidepressants

[0121] Antidepressants work by altering the concentration ofcatecholamines and/or serotonin in CNS neurons emanating from the limbicsystem into the frontal lobe. Raising levels ofcatecholamines—excitatory, adrenalin-type neurotransmitters—causesstimulation. Elevating serotonin, an inhibitory neurotransmitter,produces a calming action, and results in subsequent upregulation ofcatecholamine systems as a mechanism of habituation.

[0122] The selective serotonin reuptake inhibitors (SSRIs) inhibitreuptake of serotonin without significantly affecting adrenergicsystems. The adverse effect profiles are much less than the tricyclics,since muscarinic, histaminic and adrenergic binding is much reduced.

[0123] The effect of these serotonin agents on mood has led to morecomplex theories of how antidepressants work. According to onehypothesis, the noradrenergic systems (dopamine, noradrenalin) whichunderlie the serotonin systems respond to an increase in the inhibitoryserotonin function by upregulating, or increasing the number ofreceptors on the individual post-synaptic neural surface. This increasein adrenaline-type neural function might then account for theantidepressant activity which is delayed from the onset of serotoninreuptake inhibition by several weeks. Another possibility is that theserotonin receptors take a while to register the excess serotonin andresopond with similar mechanisms. In addition, recent evidence suggestsinteraction with DNA through transcriptases, increasing production ofneurotransmitter by producing more synthase enzymes, for example.

Antihistamines, Antiasthmatics & Histamine Agonists

[0124] Antihistamines are compounds that block histamine from activatinghistamine receptors. Since histamine functions to mediate allergicresponse, blocking histamine at H1 receptors stops the body'scharacteristic responses, i.e. inflammation and vasoconstriction. H2receptors in the stomach regulate the release of gastric acid; hence thenew class of H2-blockers such as Zantac and Tagamet stop the secretionof acid by selectively blocking these receptors without affecting the H1receptors responsible for allergic response.

[0125] Histamine is concentrated in mast cells, cells whose function isessentially to release histamine and immunoglobins when tissue damageoccurs. Receptors on the cell surface trigger lysing (breaking open) ofthe cell, releasing these allergic mediators. Mast cells are especiallynumerous in parts of the body that are injured often, such as thefingers and toes, or which enjoy frequent contact with the environment,such as the mucosa of the lips, nose, etc.

[0126] Histamine is also a neurotransmitter in the CNS and a typicalproblem with some antihistamines is drowsiness. The effort has been toproduce compounds that do not enter the brain very well.

[0127] Fenethazine represents a tricyclic antihistamine very similar toThorazine, a strong antipsychotic dopamine blocker. Cyproheptadine(Periactin), which also acts at serotonin receptors, resembles thenow-popular Claritin. Periactin has been prescribed psychiatrically foranxiety. Benadryl is probably the most familiar of the class; it hasstrong sedating qualities. Hydroxyzine also has been prescribed as asedative. Dimenhydrinate has been marketed as an anti-nausea medicationas Dramamine. Cetirizine (Zyrtec) is a metabolic product of hydroxyzine;since hydroxyzine is available as a generic, it is cheaper than thenewer drug and just as effective.

[0128] Azelastine (Astelin) has a novel structure but also acts on bothH1 and H2 receptors. Cromolyn works by a distinct mechanism; it preventsrelease of histamine following immunoglobin binding on mast cells(prevents mast degranulation).

[0129] H2-selective antihistamines have become popular as treatments forexcess stomach acid. These histamine blockers are very similarstructurally and mechanistically. All four have about the samebioavailability, half lives, and antagonist activity.

[0130] Omeprazole (Prilosec) and lansoprazole (Prevacid) belong to aclass of antisecretory compounds, the substituted benzimidazoles, thatdo not exhibit anticholinergic or histamine H2-receptor antagonistproperties, but that suppress gastric acid secretion by specificinhibition of the (H+,K+)-ATPase enzyme system at the secretory surfaceof the gastric parietal cell. These proton pump inhibitors have emergedas a therapeutic alternative to histamine antagonists for the treatmentof gastric disorders, especially acid reflux disease (“heartburn”).

[0131] For any receptor system it is possible to find drugs that act asmimetics or agonists. With histamine receptors these drugs do not findmuch clinical usefulness, but it is gratifying—to some!—to enumeratethem nevertheless. 2-methylhistamine acts as a selective agonist at H1receptors; 4-methylhistamine is relatively selective at H2. Impromidineantagonizes H2 receptors but functions as an agonist at H3.

Antiasthmatics

[0132] Asthma is essentially an accentuated allergic response to theenvironment, i.e. an autoimmune disorder. The process of allergicresponse is complex, which gives one many points at which to attack theproblem. First, immunoglobin and antigens bind to the surface of mastcells. Mast cells then release histamine, leukotrienes, peptides, whichbind to tissue receptor sites and modify intracellular chemicalprocesses governing various functions such as blood pressure regulation,smooth muscle tone, fluid disposition, etc. Compounds that inhibit anyof these steps can be used to treat asthma and allergy, beginning withthe antihistamines listed above.

[0133] Perhaps the oldest method for reducing asthma symptoms isbronchodilation by methylxanthine compounds like caffeine andtheophylline. These are currently outmoded by other compounds thatperform the same function more selectively like the beta2-adrenergicagonists (beta agonists). Methylxanthines act by inhibiting the enzymewhich effects cAMP degradation (phosphodiesterase) and by antagonizingadenosine, which causes bronchoconstriction. The beta agonists, likemetaproterenol, isoetharine, isoproterenol, terbutaline, and albuterol,mimic adrenaline at a subset of adrenaline receptor which controls thetone of smooth muscle like that of the bronchi. Another older class ofdrugs, antimuscarinics, exemplified by atropine, has enjoyed somehistorical use.

[0134] Zafirlukast (Accolate) represents a line of attack on theleukotriene compounds released along with histamine from mast cells.Leukotrienes are mediators like histamine which bind to receptors ontissue cells to signal an allergic reaction. Blocking them blocks thesignal to the (bronchial) cells and thus the undesirable response.

[0135] The final mechanism one can attack is the slow inflammatoryresponse to binding by leukotrienes and/or histamine. The body regulatesinflammation with glucocorticoid steroids, and synthetic compounds suchas fluticasone (Flonase) and beclomethasone (Beclonase) are effectivemimetics.

Antihyperlipidemics

[0136] Antihyperlipidemics are relaively new drugs which lower bloodcholesterol levels and help to prevent atherosclerosis by inhibiting theformation of plaque on arterial and vascular linings. The formation ofthis plaque is dependent on the proportion of various types ofblood-fats, particularly on the ratio of high-density lipoproteins(HDLs) to low-density lipoproteins (LDLs). This proportion is in turninfluenced by genetics and by the amount of certain substances in thediet, particularly cholesterol and saturated fat. Cholesterol isessential in the formation of VLDLs, large lipoproteins produced by theliver; on catabolysis by lipoprotein lipase, VLDLs produce the smallerLDLs, which are the so-called “bad cholesterol,” HDLs being termed “goodcholesterol” in the common parlance. Because the production of theselipoproteins is complex, several points can be targeted for action byvarious drugs.

[0137] The most commonly used antihyperlipidemics are simvastatinanalogs, especially simvastatin (Zocor) itself. These drugs, known asHMG-COA reductase inhibitors, decrease production of cholesterol byinhibiting the first step in sterol synthesis which involves productionof mevalonate ion from CoA-S-mevalonate. Decreased cholesterol formationresults in a reduction in VLDLs and hence LDLs. Pravastatin is theactive metabolite of mevastatin; lovastatin and simvastatin are inactiveprodrugs that work through their hydroxyl derivatives, obtained bysimilar ring-opening.

[0138] These drugs also stimulate receptor-mediated clearance of LDLs.LDL receptors undergo upregulation (increase in receptor density) andVLDL catabolysis is increased.

[0139] Recent studies suggest a reduction in osteoporosis as a result oftreatment with statin drugs.

[0140] Other drugs like clofibrate and gemfibrozil increase the activityof lipoprotein lipase, reducing the level of VLDLs. This enzyme is anextracellular species present in the blood and gut. Effects on otherlipase enzymes may account for the nausea common to this drug.Cholesterol production in the liver is reduced, probably as a secondaryeffect due to lower VLDL levels, while fecal cholesterol excretion isenhanced. Clofibrate is fairly toxic and may be carcinogenic.

Antihypertensives

[0141] The body controls blood pressure by a complex feedback mechanismbetween baroreceptors and effector nerves, primarily adrenergic innature. This system is modulated by a peptide systems(angiotensin/renin).

[0142] Pharmacologic control of blood pressure acts through four basicmechanisms. Sympathoplegics reduce peripheral vascular resistance,inhibit cardiac function and increase venous pooling by a number ofmechanisms involving adrenergic nerves.

[0143] Direct-acting vasodilators decrease blood pressure by increasingblood volume. Veins and arteries are forms of smooth muscle, andrelaxing the muscle results in larger volume and lower pressure.Angiotensin antagonists work on peptide systems with effects on smoothmuscle. Finally, diuretics decrease sodium content, decreasing bloodvolume and thus blood pressure. Because the body responds to exogenousagents by homeostatic regulation, concomitant use of several agentsworking on different mechanisms is frequently used, rather than simplyincreasing the dose of a single med.

[0144] Sympathoplegics or sympatholytics antagonize the function ofadrenalin compounds. Beta blockers block beta-1 receptors in hartmuscle, decreasing cardiac output. The include propranolol (Inderal),metoprolol (Lopressor), labetolol (Normodyne), nadolol (Corgard),atenolol (Tenormin), and acebutolol (Sectrol). Someadrenoceptor-activating compounds have a hypotensive effect by actingcentrally at alpha receptors. These include clonidine (Catapres),guanfacine (Tenex), guanabenz (Wytensin), and methyldopa. These probablywork by activating presynaptic autoreceptors, decreasing norepinephrine(agonist) release.

[0145] Compounds that block alpha-1 receptors peripherally, in bloodvessels, include prazosin (Minipress) and terazosin (Hytrin).

[0146] Reserpine (Serpasil) blocks amine uptake, while guanethidine(Ismelin) and guanadrel (Hylorel) block sympathetic nerve terminals.

[0147] Propranolol, which was formerly the most prescribed drug of theclass, leads to accumulation of bradykinin, which contributes to theantihypertensive effect.

[0148] Some anticholinergic drugs (ganglion blockers) are effective ashypotensives, but they are less popular because of side effects. Theseinclude trimethapan (Arfonad) and mecamylamine (Inversine).

[0149] Diazoxide acts by opening potassium channels and relaxing smoothvenous and arterial muscle. Despite structural similarities, it has nodiuretic properties. It has a relatively long half-life of about 24hours, and is often used parenterally (by injection) in emergencies. Thedrug also inhibits insulin release and is used in diabetes. Minoxidil ismainly used orally, also opening potassium channels.

[0150] Hydralazine and minoxidil dilate arterioles but not veins.Nitroprusside (as the sodium salt) dilates both, by a mechanisminvolving activation of guanylyl cyclase, resulting in formation of cGMPand relaxation of smooth muscle.

[0151] The calcium channel blockers are a popular means to controlhypertension. Smooth muscle contraction depends on calcium influx tocontrol muscle tone. The reaction path is complex, involving thepeptides calmodulin and myosin light chain kinase (MLCK). When thelatter enzyme is activated it phosphorylates myosin which acts withactin to contract muscle. Blocking the channel statistically preventscalcium ion influx and decreases tension in blood vessel smooth muscle.Skeletal muscles rely on intracellular calcium ion, and are not affectedby these drugs. Cardiac muscle is highly dependent on calcium channelaction.

[0152] Calcium channels also exist at presynaptic nerve terminals inadrenergic neurons. These are voltage-dependent ion channels embedded innerve membranes at the ends of adrenergic neurons. When a voltage pulsearrives at the end of a neuron (propagated by sequential firing ofsodium channels), the calcium channels detect the change in voltage andallow influx of Ca⁺⁺ ions. This triggers binding of vesicles and releaseof vesicular adrenalin, noradrenalin or dopamine, along withcotransmitters such as peptides, ATP, etc. One of the acid tests as towhether a substance is a neurotransmitter is whether its release iscalcium-dependent.

[0153] Verapamil is the oldest and prototypical calcium channel blocker.It is highly bound to blood plasma proteins and suffers about an 80%hepatic first-pass elimination on oral administration. This means thatmost of the drug absorbed through the intestine is removed by the kidneybefore reaching the target tissues (heart and major blood vessels).Nifedipine is significantly less active at cardiac sites than diltiazemor verapamil, and is also highly plasma-bound. Diltiazem is much lessplasma bound. Despite plasma binding, all three drugs have fairly shorthalf-lives (3-6 hours).

[0154] Ion channel blockers generally act by lodging in an open channeland blocking it. Similarities in ion channels mean that some sodiumchannel blockade occurs with calcium channel blockers. This is more of aproblem with verapamil than the other drugs. In addition to treatment ofhypertension, these drugs are used to treat angina, migraine, andatherosclerosis.

[0155] Diuretics reduce blood volume by causing excretion of waterthrough the kidney. This reduces blood pressure very effectively. Thedrugs used for this purpose are typically thiazides; a main problem ispotassium depletion. Potassium-sparing diuretics have also beendeveloped.

[0156] Recent studies have shown the ACE inhibitors to be extremely safedrugs. ACE is an enzyme which converts angiotensin I to angiotensin II,the active form. Angiotensin receptors modulate the tension of smoothmuscle, including venous and arterial tissue. Inhibiting the enzymedecreases the amount of active peptide extant in body tissues.

Antipsychotics, Lithium, Mood Stabilizers & Dopamine Agonists

[0157] Antipsychotics are used to calm mania or racing thoughts, tocontrol aggression, or to block spurious thoughts in schizophrenia,including auditory hallucination (hearing voices). They exert theirtranquilizing effects by blocking the excitatory neurotransmitterdopamine at post-synaptic terminals. Dopamine neurons are abundant inthe limbic system and its projections into the cerebrum, especially theoriginating in the substantia nigra. Blocking of adrenergic, histaminicand serotonergic receptors also contribute to the CNS effects of thesedrugs.

[0158] Although they are potent psychotropics, these drugs are notcommonly abused, since they inhibit the brain's pleasure pathways whichemanate from the limbic system into the frontal lobe.

[0159] Tricyclic compounds like Thorazine, known as phenothiazines, arethe oldest compounds, and are the least selective, blocking severalsubtypes of dopamine receptors. Thorazine was discovered by accidentwhile seeking better antihistaminic agents. It has shown efficacy inblocking the effects of LSD, confirming the dopamine agonist activity ofthat drug.

[0160] Newer compounds of the haloperidol class (bytyrophenones), firstsynthesized in the late 1950s, are more selective for D2 subreceptors.

[0161] Clebopride and sulpiride analogs represent another structuralclass of antipsychotics with similar actions. The binding profile of allthese groups (indeed, of each compound) will be slightly different.

[0162] The clozapine analogs represent another structure type with adifferent neurological profile. They are not as selective for D2 as thehaldol class, but may be more effective at controlling some types ofpsychosis. These drugs show significant 5-HT2 receptor blockade and maybe more selective for limbic dopamine systems (as opposed to thoseinvolved in motor control), reducing the extrapyramidal syndrome (EPS)and dyskinesias common to antipsychotic meds. The newer agents, such asZyprexa and Seroquel, seem not to impart the agranulocytosis common withclozapine.

[0163] Clozapine is extensively metabolized and some of its metabolitesshow anti-AIDS activity.

[0164] Other novel structural classes which function as neuroleptics arerepresented by butaclamol (a pentacyclic) and tetrabenazine. The latteris a dopamine depleter, a drug which can chemically induce depression.

[0165] The use of anticonvulsant medications for psychotropic purposeshas recently grown, primarily to prophylact against manic and/or panicsyndromes. Phenytoin and carbamazepine probably work by affectingion-gating systems (particularly sodium channels) in excitablemembranes. Phenytoin structurally resembles the barbiturates whilecarbamazepine has a tricyclic structure like the tricyclicantidepressants.

[0166] Gabapentine and valproic acid probably work on GABA systems. Thelatter inhibits the enzyme responsible for degrading GABA at highconcentrations, but probably works by other mechanisms at lower,therapeutic levels. Lamotrigine is another antiepileptic used as a moodstabilizer. It reduces release of glutamate, an excitatory amino acid.Topamax, a fructose derivative, enhances GABA systems while blockingglutamate.

[0167] Propanolol is a beta-adrenergic blocker prescribed forperformance phobia or stage fright. Clonidine is an alpha agonistsometimes used to calm peripheral tremor as in alcohol withdrawal.Verapamil, a calcium-channel blocker, is also used for this purpose.

[0168] Pergolide and naxagolide are dopamine agonists employed againstParkinsonism, which results from decreasing dopamine function in the CNSwith aging. Apomorphine is a selective D2 agonist, while ibopamineserves agonist function at both dopamine and adrenergic receptors.

Cholinergic Drugs

[0169] Acetylcholine neurons convey sensory information to the brain andcontrol muscular tension, including peristalsis and motor control.Cholinergic neurons are dominant in inhibitory activity inherent toso-called parasympathetic neurons whic comlpementdopamine/norepinephrine based neurons in parallel sympathtic structures.Two cholinergic receptor subtypes have been identified by selectiveagonists: nicotinic and muscarinic. At least two subtypes of muscarinicreceptors (M1 and M2) have been identified.

[0170] In addition to direct agonists, selective antagonists, enzymeinhibitors, and antidotes to enzyme inhibitors have been developed.Cholinoceptors also serve as heteroreceptors, presynaptically governingthe release of norepinephrine and other neurotransmitters.

[0171] Nicotine is a selective agonist at nicotinic receptors: itdefines this subset of cholinergic receptors. Muscarine defines theother subset, with further distinctions of M1 and M2 (at least)existing. Muscarine is produced in trace amounts in the fly agaricmushroom. Other species of fungus produce greater amounts. Fly agaricalso contains muscarinic antagonists (atropine) and GABA agonists(muscimol). Atropine used to be applied as an antidote to poisoning bymuscarine in this fungus, before the role of muscimol was elucidated.

[0172] The N-hydroxymethyl amide of nicotinic acid is also active as anagonist at nicotinic cholinoceptors. Carbachol is used opthalmically asa miotic, i.e. to dilate the pupils. It is also used in large animals,mainly in atonic conditions of the gut, since its formal positive chargeprevents it from entering the brain and limits its absorption in thegut. In addition to receptor action, it probably promotes acetylcholinerelease. Lachesine is a selective muscarinic agonist.

[0173] Guanidine exists as the guanidium ion at physiologic pH; it isused as a pro-cholinergic, antiviral, antifungal, antipyretic and musclestimulant. Bethanechol activates M1 and M2 subreceptors, releases IP3(inositol triphosphate), and activates guanylyl cyclase. Again, as aquaternary, positively charged species, it is used mainly to mimicacetylcholine in the gut. It is sometimes given to relieve theantimuscarinic constipation caused by tricyclic antidepressants or othermeds. Pilocarpine is a cholinomimetic which also increases gastric acidsecretion.

GABA Drugs

[0174] GABA (gamma-aminobutyric acid) is the most important inhibitoryneurotransmitter in the CNS. By gating negative chloride (Cl−) ions intothe interior of nerve cells, GABA inhibits the presynaptic release ofneurotransmitter due to a positive voltage polarization pulse. Suchinhibition is extremely common: GABA receptors can be found at 60-80% ofCNS neurons.

[0175] Subtypes of GABA receptors can be activated by the mushroom toxinmuscimol (at the A subtype) as well as the antispasmodic amino acidbaclofen (B subtype). These drugs directly mimic the action of GABA atthe receptor.

[0176] Allosteric facilitation of GABA receptors occurs at severaldistinct sites; the compounds which bind there are used as sedatives andanxiolytics. These compounds bend the receptor open to indirectlyfacilitate GABA binding.

[0177] Progabide is a pro-drug which decomposes to GABA in the CNS. Itcrosses the blood-brain barrier, which GABA itself, being a zwitterion(doubly-ionized amino acid), does not. Vigabatrin (gamma-vinyl-GABA)inhibits GABA-aminotransferase (GABA-T), the enzyme responsible fordegrading GABA in the synapse. It thus prolongs the sojourn of GABAmolecules and promotes binding in this way.

[0178] Depakote (valproic acid) seems to act on nerve membranes toreduce susceptibility to seizure. At high doses it acts like vigabatrinto inhibit GABA-T. Gabapentine is another recently marketedantiepileptic (Neurontin) that is also finding psychiatric applicationas a mood stabilizer. The neurological rationale for this application isthat panic attacks (or mania in bipolar disorder) resemble epilepsy inthat they are characterized by a pre-panic “kindling” phenomenon,characterized by repetitive neural firings, leading to a critical stage.Gabapentine may encourage production of or discourage degradation ofGABA. Lamotrigine probably works by reducing release of glutamate, anexcitatory neurotransmitter usually governed by the inhibitory GABA.

[0179] Novel GABA drugs represent one of the most active areas ofpsychotropic research. Riluzole, for instance, is a GABA uptakeinhibitor with anticonvulsant and hypnotic properties; it also blockssodium channels and inhibits glutamate release.

Opiate Narcotics

[0180] Opiates, derived from the poppy plant, contain alkaloids whichactivate the brain's endogenous endorphin receptors to produceanalgesia, euphoria, and respiratory suppression. Poppy opiates possessa polycyclic phenanthrene nucleus with various substituents thatdetermine the fit into the receptor. Although morphinelike compoundshave been found in mammalian brain tissue, it is generally agreed thatthe enkephalins and endorphins represent the endogenous compounds whichpoppy constituents mimic.

[0181] Opiate receptors of several varieties are responsible for themajor pharmacologic effects. These subtypes are given Greek names likemu (analgesia, euphoria), sigma (dysphoria, cardiac stimulation), kappa(sedation, spinal cord analgesia, miosis), delta, etc. Antitussiveproperties, emesis (vomiting), and anticholinergic (constipation)effects also occur, indicating a wide variety of receptor types andactions. The sigma receptor is now surmised to be related to glutamatefunction.

[0182] Opiate receptors exert effects on synaptic transmission bypresynaptically modulating the release of neurotransmitters, includingacetylcholine, norepinephrine, dopamine, serotonin, and substance P. Thelatter compound is a peptide neurotransmitter involved in nociceptive(pain-related) neurons. Opiate receptors act on G-peptides,transmembranal macromolecules linked to post-synaptic intracellularenzymes (such as adenylyl cyclase) or ion channels (such as K⁺, Ca⁺⁺).In high doses the opiates cause generalized CNS depression sufficientfor surgical anesthesia.

[0183] Codeine is a mild analgesic which retains agonist activity atother receptor subtypes including those controlling respiration,peristalsis and euphoria. Morphine is among the most potent of thephenanthrene class. The less amphoteric heroin crosses the blood-brainbarrier more readily but decomposes into morphine once there. Oxycodone,the main active constituent in Percodan and Percocet, is somewhat lesspotent.

[0184] Meperidine (Demerol) is a synthetic drug that has approximatelythe same analgesic activity as morphine. Methadone, invented by theNazis and originally named dolophine, is famous for its use in assuagingthe heroin withdrawal syndrome. Its half-life is substantially greaterthan that of heroin, and while it is bound to receptors it blocks newlyadministered heroin. Its analgesic activity is also approximately equalto morphine's, but it imparts less euphoria.

[0185] Fentanyl constitutes one of the most potent synthetics,propoxyphene (Darvon) one of the least. Methoxy compounds such ascodeine and oxycodone are less susceptible to first-pass reactions(typically conjugation to a glucuronide) and therefore have a higheroral-to-parenteral ratio. Less-amphoteric compounds (compounds with moredefinite acid or base properties) pass the blood-brain barrier moreeasily.

[0186] Alteration of the phenanthrene skeleton produces drugs with mixedagonist/antagonist properties at opiopeptin subreceptors. These drugsare being used variously as pain killers, aids in withdrawal from heroinand even alcohol addiction, and (illegally) to increase athleticstamina. Stadol has been used nasally to relieve migraines. Althoughmixed agonists retain analgesic properties, they often impart dysphoriceffects.

[0187] Narcotic antagonists are especially useful in cases of overdose,where they can reverse the CNS depression caused by opiate agonists.Naloxone is the most often used, most effective, and prototypal narcoticantagonist. Naloxone, nalmefene, and nadide are among several othercompounds used to antagonize morphine receptors.

[0188] Naltrexone has recetnly been used to reduce the craving foralcohol among recovering alcoholics and heroin addicts (as ReVia).

Nootropics & Smart Drugs

[0189] Nootropics, also known as smart drugs or cognition activators,are drugs that enhance mental function. Several mechanisms that affectnerve function may be attacked. Compounds that are used by the body tomanufacture neurotransmitters constitute one group (precursors).Reuptake and degradation inhibitors form another. Mimetics of excitatoryneurotransmitters and antagonists of inhibitory ones can both stimulateneural function. Antianoxics enhance the ability of neurons to bumglucose. Phospholipid compounds affect the fatty excitable membranes ofnerve cells, which are responsible for transporting a depolarizationpulse down dendrites and axons. Steroid compounds also affect membranechemistry. Vasodilators which act in the CNS increase blood supply tobrain cells. Still other drugs increase the flexibility of red bloodcells so they can gain access to more neurons more often. All theseeffects be theoretically be used to enhance neurological function in theCNS.

[0190] Glycine systems perform inhibitory functions in the CNS.Enhancement of these pathways imparts antianxiety effects and sostabilizes mood. Glycine itself is a zwitterion and so does not pass theblood-brain barrier very well. Dimethylglycine is stabilized by themethyl groups; its greater lipophilicity results in better transport tothe CNS, where it is converted to glycine. Milacemide is a pro compoundwhich decomposes (via MAO-B) to glycinamide and then glycine in the CNS.

[0191] Glutamate and aspartate are another group of excitatoryneurotransmitter prominent in the CNS. Since they are acidic amino acidsthey have difficulty crossing the blood-brain barrier, but standardtricks can be used to deliver them to the CNS. Making an amide out of acarboxy acid is one of these (as in glutamine and aceglutamide); asomewhat more radical method is to make a covalent salt with calcium, asin calcium-N-carbamoylaspartate.

[0192] Carnitine is a catabolic (tearing-down) amino acid which servesas a neuroprotectant at NMDA receptors (a subset of glutamate/aspartatereceptors). Acetylation of the hydroxy group gives ALC, which again hasthe effect of promoting transport into the CNS.

[0193] Several steroids have been used to bolster mental function andlibido. Both testosterone and estrogens have been administeredhistorically to increase vitality and sexual drive as people grow older(replacement therapy). Precursors to estrogen and androgen steroids suchas DHEA and pregnenolone have recently been marketed as nutrients. Thesesteroids do not have significant estrogenic or androgenic propertiesuntil converted by the body to active forms. As with all precursors, onetrusts the body's homeostatic mechanisms to regulate formation of activemolecules by rate-limiting steps, competitive mechanisms, andtachyphylaxis (tolerance).

[0194] The use of anticonvulsant medications for psychotropic purposeshas recently grown, primarily to prophylact against manic and/or panicsyndromes. Phenytoin and carbamazepine probably work by affectingion-gating systems in excitable membranes. Phenytoin structurallyresembles the barbiturates while carbamazepine has a tricyclic structurelike the tricyclic antidepressants. Propanolol, a beta-blocker, has beenused to calm peripheral reactions to stress, such as stage fright.

[0195] Propanolol is a beta-adrenergic blocker prescribed forperformance phobia or stage fright. Clonidine is an alpha agonistsometimes used to calm peripheral tremor as in alcohol withdrawal.Verapamil, a calcium-channel blocker, is also used for this purpose.

[0196] The piracetam group of antianoxic compounds work by severalmechanisms to invigorate neural function. By supplying glutamic acidanalogs to the Krebs cycle they enhance glucose utilization in aerobicrespiration, the major means by which animal cells extract chemicalenergy from sugars via ATP formation. This in turn raises phospholipidcAMP levels, enhancing the function of dopamine and acetylcholineneurons. Additionally they function as antioxidants (compare thestructure to that of vitamin C) and retard lipofuscin formation.Experimentally, piracetam has been shown to increase athleticperformance, to reverse alcohol-induces brain degeneration, and has beentried as a treatment for dyslexia.

[0197] Methylxanthines are used as bronchodilators in the treatment ofasthma (typically theophylline) and in conjunction with analgesics totreat headache. Pentoxyfylline and propentofylline have central andperipheral vasodilatory properties. Increased blood supply to braintissue probably accounts for whatever nootropic properties they have.Pentoxfylline also increases the elasticity of red blood cells, enablingthem to better squeeze through constricted capillaries. Such drugs arecalled anti-ischemics, ischemia referring to a lack of blood supply to atissue.

[0198] Pyritinol is another vasodilator which has been used againstdementia senilis (senility). Idebenone resembles ubiquinone, a compoundwhich catalyzes mitochondrial metabolic processes. It promotes secretionof nerve growth factor (NGF) and may also protect cell membranes againstlipid peroxidation. Ergocryptine is an ergot alkaloid which has beenused to combat age-related memory loss and Alzheimer's. It dilates bloodvessels by blocking alpha-adrenoceptors. It has been used in accidentvictims to increase blood flow to the brain following trauma to preventtissue damage by anoxia. Vinpocetine and vincamine are two alkaloidsfrom the vinca plant which also have anticoagulant and vasodilationeffects.

Non-Steroidal Anti-Inflammatory Drugs (NSAIDS)

[0199] Nonsteroidal anti-inflammatory drugs (NSAIDS) are the drugs ofchoice for mild to moderate pain and to reduce fever (antipyretics).They interfere with the formation of prostaglandins by inhibiting theenzyme cyclooxygenase, which closes a bond on arachidonic acid, anessential oil. The cyclical prostaglandin compounds are potent,short-lived mediators of the inflammation response. They are not storedin cells but are synthesized as needed in response to injury orirritation. Interfering with their production peripherally turns off theinflammation response in the body.

[0200] Aspirin and the other NSAIDs may also act at a site in the CNS.Some of the NSAIDs (e.g. ketoprofen) inhibit other enzymes such aslipoxygenase, further retarding the allergic/inflammation response.

[0201] Aspirin (acetylsalicylic acid) has been used for centuries alongwith its relative, methylsalicylate. The latter, known as oil ofwintergreen and often used topically, is even more toxic than aspirin inoverdose. Aspirin interferes with platelet aggregation and retardscoagulation of blood. This property probably accounts for its use in thelong-term prevention of heart attacks.

[0202] In recent years drugs like ibuprofen and ketoprofen have becomeavailable over-the-counter. The more lipophilic of these drugs, such asnaproxen (Aleve, Naprosyn), ketoprofen (Actron) and nabumetone(Relafen), have longer half-lives, requiring less frequent dosing, butare probably no more effective analgesics than ibuprofen. Liver, kidneyand GI problems, of varying seriousness commensurate with dosagehistory, are common. Penicillamine has been used as a long-acting NSAID,but it is fairly toxic, causing reduction of healing and a host ofautoimmmune and histological disorders.

[0203] A new class of NSAIDs inhibit cyclooxygenase-2, an enzymeresponsible for interconversion of prostaglandins. These COX-2inhibitors are intended to preserve the formation of cytoprotectiveprostaglandins while targeting inhibition of the compounds responsiblefor pain and inflammation, reducing stomach irritation, Celebrex(celecoxib) is one such drug. Vioxx is another new drug in this class.

[0204] Recent studies have suggested that a pair of aminated sugarcompounds can assist in repairing damage to cartilage in osteoarthritis.Glucosamine, a monomer, and chondroitin, a polymer, are being marketedas nutrients for this purpose. In cartilage, sugar polymers form aflexible connecting matrix around the tough protein strands in cartilage(a composite material).

[0205] The benzodiazepine sedatives include Valium, Librium, Halcion,Xanax, Ativan, Serax, and Klonopin, to name just a few. In addition topotential effects on lipophosphate nerve membranes, these drugs work byallosterically enhancing the effect of the inhibitory neurotransmitterGABA at post-synaptic receptors. That is, they “bend” the receptorslightly so that GABA molecules attach to and activate their receptorsmore effectively and more often. Their chief advantage over thebarbiturates, such as seconal, nembutal and phenobarbital, is that theydo not act directly to open chloride ion channels.

Serotonin Drugs

[0206] Serotonin is an inhibitory neurotransmitter which complementsexcitatory sympathetic systems like adrenaline and dopamine in the CNS.Like the “fight or flight” adrenaline compounds, serotonin is releasednot only at specific synaptic sites, but also in a broadcast manner intobrain tissue from sets of “diffuse” neurons emanating from the emotionalcenters in the limbic system into the frontal lobe. This diffuse releasesets the biochemical tone of large areas of neural functioning,controlling mood and motivation. Serotonin's inhibitory action ishowever more complex and selective than that of GABA sedatives likeValium or Xanax, which act more globally. Because of their effects onmood, serotonin-active drugs are used as antidepressants and anxiolytics(anti-anxiety) drugs.

[0207] Ondansetron is a selective 5-HT3 antagonist. This receptorsubtype is found on cholinergic neurons; when it is activated itinhibits release of acetylcholine. Along with its chemical relativessuch as granisetron and zatosetron, it may thus be useful in revivingmemory function in the aged. Granisetron is also used as an antiemetic(Kytril) in chemotherapy.

[0208] Ketanserin, a selective 5-HT2 antagonist, also acts on alpha-1adrenoceptors to lower blood pressure. Mescaline, a hallucinogen,antagonizes 5-HT2 terminals and has been tried as an alternative todopamine blocking antipsychotics (without much success; it facilitatesdopamine function). Oxetorone is a relatively new antagonist usedagainst migraine, as is pizotyline. Cyproheptadiene is an olderserotonin antagonist and antihistaminic. Mirtazapine (Remeron) causesserotonin release, but blocks the 5-HT2 and 5-HT3 subreceptors,effectively augmenting serotonin action at 5-HT1 receptors. Mianserinand homochlorcyclizine also antagonize serotonin receptors.

[0209] Sumatriptan activates 5-HT1d terminals, and is used againstmigraine under the trade name Imitrex. Zolmitriptan (Zomig) andrizatriptan (Maxal) are similar, recently approved, antimigraineserotonin drugs.

[0210] Buspirone, ipsapirone and gepirone enjoy 5-HT1 agonist propertieswith only weak D2 blocking effects. Buspirone is used against anxiety asan alternative to GABA-mimetic sedatives. 8-hydroxy-DPAT actsselectively at 5-HT1 a receptors, while 2-methylserotonin activates5-HT3 terminals.

Steroids & Reproductive Drugs

[0211] Steroids are fat-soluble (lipophilic) hormones with a tetracyclicbase structure. The steroid structure is synthesized from smallerstructures called terpenes to precursor molecules, which then undergoextensive and subtle alterations for a rich variety of uses: to controlsystems, often in fatty tissues, as diverse as meiosis, carbohydratemetabolism, fat storage, muscle growth, immune function, and nerve cellmembrane chemistry. Because of their high lipophilicity, they can passthrough cell membranes, which are fatty bilayers, and influence DNAtranscription and thereby alter protein synthesis. By binding tospecific sites on the DNA, they release a kind of molecular “boot”(hsp90) that, in the absence of steroid, locks up the DNA and prevents ashort sequence from being expressed into a protein. The action of theenzymes or active peptides generated by the activation of the DNA canpersist for long periods of time, explaining the long duration of actionof many steroids.

[0212] Steroids may be separated into the broad groups of gonadalcompounds and glucocorticoids, depending on the site of synthesis, whichis in the ovary or testis for the gonadal variety and in the adrenalcortex for glucocorticoids. They may also be divided according tofunction, with the usual designations being androgens, estrogens, andprogestogens (typically for the gonadal hormones) and anabolics andcatabolics (typically for the glucocorticoids).

[0213] It is important to realize, however, that these terms are nottotally exclusive. That is, even the gonadal hormone testosterone issynthesized in small quantities by the adrenal gland, and impartsanabolic properties separate from its effects on gonad function.Moreover, all hormones act in coordination with other compounds toproduce a net result.

[0214] Testosterone is the prototype of the androgen group of gonadalsteroids. Androgens impart features typified by males of mammalianspecies. These include morphological features such as a protrudingbrowridge, robust bone structure, and large canines. It is alsoresponsible for aggression and libido. It also acts as an anabolic,aiding muscle formation in response to exercise. Estradiol, meanwhile,is the prototypical estrogen, imparting female characteristics such asbreast growth and storage of subcutaneous fat. Estrogens also preventheart disease (women get it statistically less than men).

[0215] Oral contraceptives reformulate the body's steroid chemistry tomimic that of pregnancy to prevent ovulation. This is usually done inaccordance with the natural 28 day menstrual cycle, although it ispossible to trick the body into delaying ovulation for longer periods.This is accomplished by a mixture of an estrogen and a progestogen. Themost popular preparation is norethindrone (a progestogen) andethinylestradiol (an estrogen). This combination, taken in a large dosejust after unprotected sex, can also prevent pregnancy by the samemechanism. Replacement therapy for gonadal steroids in the form oftestosterone for men and estrogen/progesterones (depot ProVera) andrecently also testosterone for women has been tried to combat thesymptoms of aging, including diminished sex drive. In men, testosteronehelps libido and may improve cardiovascular fitness and general vigor.In women, the drop in estrogen after menopause imparts some changes, butthe drop is relatively modest (20% or so) compared to the drop inprogesterone, which causes osteoblasts to make new bone tissue andinhibits cancer cell formation. Since estrogens, being anabolic ortissue-building compounds, can promote cancer cell growth, modemreplacement formulations should compensate more for progesterones thanestrogens. Non-steroidal soybean estrogens are now marketed astreatments for menopausal hot flashes, and testosterone creams andtablets to increase sex drive.

[0216] Cholesterol is heavily involved in membrane in metabolicchemistries. One of its main uses in the body is to decrease thepermeability of phospholipid cell walls to ionic species such as Na⁺,K⁺, and Ca⁺⁺. In recent years it has been implicated in aiding theaccumulation of plaque on the interior surfaces of veins and arteries.Animal meat is typically rich in cholesterol. Through natural selection,carnivores such as cats and dogs have developed different chemistriesadjusted for processing higher amounts of cholesterol, cholestanol (thesaturated from), and other steroids, which make diets higher in meatsafer for them than for us. Cholesterol is excreted through gall, afatty digestive substance secreted from the liver. It is present in highquantities in gallstones. Bile is also used to excrete fat-solublesubstances such as bilirubin (from the heme group in decomposed redblood cells). Cholesterol is also used endogenously to synthesizevitamin D.

[0217] Cortisone, another prototypical glucocorticoid, controls healingprocesses associated with the immune system, as well as regulatingmembrane and other functions. Hydrocortisone, also known as cortisol,works similarly to inhibit histamine-mediated allergic reaction andregulates the body's response to stress by modulating the chemistry ofneuronal excitable membranes. Prednisone is a synthetic compound usedregularly in place of cortisone. Dexamethasone has been used to diagnosedepression, i.e. in the dexamethasone suppression test, where the bodyis “stressed” by introduction of the steroid and its response ismeasured.

[0218] Hexestrol and diethylstilbestrol are two examples of polycyclic,non-steroid compounds which activate estrogen receptors. The localstructure in the bound configuration resembles that of steroids. Theother main category of non-steroidal estrogens is the isoflavones.

[0219] Environmental estrogens have become a health concern since cattleare commonlty fed estrogens due to their anabolic (weight-gain)properties. Ingestion of meat therefore equates to absorbing someestrogens. Some pesticides are non-steroidal estrogens, as is THC, themain psychoactive constituent of marijuana. Compounds such asdiethylstilbestrol have been shown to be carcinogens, though this is notdue to action on DNA.

[0220] Recently, two nonsteroidal estrogen agents have shown greatmedical promise in several women's health issues. These selectiveestrogen receptor modulators (SERMs) mimic the effects of estrogens insome tissues but not others.

[0221] Tamoxifen has been used for years following detection of breastcancer. By blocking estrogen receptors, it discourages tumor growth. Newstudies show it may also prevent breast cancer, probably by the samemechanism. However, the benefits of this prevention must be weighedagainst the increased risk of uterine cancer and other potential risks.

[0222] Raloxifene retains the ability to promote bone maintenance andprevent osteoporosis; it cuts the risk of breast cancer by as much as60%, and decreases levels of LDLs (“bad” cholesterol).

[0223] Finasteride (Proscar, Propecia) is presently being marketed as asystemic (oral) anti-baldness medication with a mechanism distinct fromthat of minoxidil, which is a topical vasodilator which stimulatesfollicular activity by improving blood flow. Finasteride works byinhibiting the formation of 5-alpha-dihydrotestosterone, a potentandrogen, from the less potent parent compound, testosterone. It hasalso been used to treat benign prostatic hypertrophy (enlargement of theprostate).

[0224] The side effects of reducing androgens in the body can beessentially termed feminization: atrophy of the male gonads, breastaugmentation, decrease in aggressive behavior, increased risk ofosteoporosis, etc.

[0225] Steroids are not confined to the animal kingdom but aresynthesized by plants as well. The well-known cardiotonic digitalis isderived from the foxglove plant which synthesizes several glucosidesteroids (i.e. steroids bonded to sugar moieties).

[0226] The oleander shrub generates several steroids with similareffects on cardiac conduction, including oleandrin and oleandrigenin.

[0227] Sildenafil (Viagra) is a erection facilitator. Erection dependson an interaction of adrenergic and cholinergic neurons in which musclesmust relax to let blood into erectile tissue. The presence of nitrousoxide (NO) species is involved, and Viagra may affect the enzymesresponsible for generating NO. Viagra is also a selective inhibitor ofcGMP phophodiesterase, which acts in some GI vascular smooth-muscles.Caffeine, wose central ring structure resembles Viagra's, workssimilarly on the more widespread cAMP phosphodiesterase, a morewidespread second messenger system. Yohimbine, a selective alpha2adrenergic blocker, has also been touted as being able to prolong orintensify erection.

[0228] Gossypol, isolated from the cotton plant, has the ability toinhibit production of viable sperm in men. It damages the epitheliallining of seminferous vesicles, inhibiting sperm formation. It alsopoisons the oxygen-carrying capacity of blood.

[0229] Among peptide therapeutic candidates are peptides whichdemonstrate anti-neoplastic activity, such as the RGD peptides includingpeptide SEQ ID NOS. 1-3.

[0230] Also included are peptides that are active against HIV, includingthe GP-41 peptides including peptide SEQ ID NOS. 4-6.

[0231] Anti-viral peptides which demonstrate the ability to disruptfusogenic events common in viral infections. These include the RSVpeptides which demonstrate the ability to treat or prevent infection byrespiratory syncytial virus (RSV) as well as acquired immune deficiencysyndrome (AIDS) caused by infection of the human immunodeficiency virus(HIV). Such peptides include peptide SEQ NOS. 7-9.

[0232] Also included are GLP-1 peptides including those peptidesdepicted in SEQ ID NOS. 10-11.

[0233] Also included are Kringle or K5 peptides including those peptidesdepicted in SEQ ID NOS. 12-13; BBB peptides (TAT) including thosepeptides depicted in SEQ ID NOS. 14-15 and analgesic peptides, such asdynorphins, are also useful, including peptide SEQ ID NO. 16.

[0234] 3. Modified Therapeutic Agents

[0235] The modified therapeutic agents of the present invention comprisetherapeutic agents that have been modified by attaching a reactivegroup. The reactive group may be attached to the therapeutic agent via alinking group, or optionally without using a linking group. The modifiedtherapeutic agents can react with the available functionalitieson bloodor pulmonary components or blood components via covalent linkages. Theinvention also relates to such modifications, such combinations withpulmonary components or blood components, and methods for their use.These methods include extending the effective therapeutic life of theconjugated therapeutic agents as compared to administration ofunconjugated therapeutic agents.

[0236] To form covalent bonds with functionalities on a protein, one mayuse as a reactive group a wide variety of active carboxyl groups,particularly esters, where the hydroxyl moiety is physiologicallyacceptable at the levels required to modify the therapeutic agent. Whilea number of different hydroxyl groups may be employed, the mostconvenient would be N-hydroxysuccinimide (NHS),N-hydroxy-sulfosuccinimide (sulfo-NHS), maleimide, maleimide acidsincluding but not limited to maleimidopropionic acid (MPA), andmaleimide esters. In the preferred embodiments of this invention, thefunctionality on the blood component will be a thiol group and thereactive group will a maleimide.

[0237] Primary amines are the principal targets for NHS esters.Accessible α-amine groups present on the N-termini of proteins reactwith NHS esters. However, α-amino groups on a protein may not bedesirable or available for the NHS coupling. While five amino acids havenitrogen in their side chains, only the ε-amine of lysine reactssignificantly with NHS esters. An amide bond is formed when the NHSester conjugation reaction reacts with primary amines releasingN-hydroxysuccinimide as demonstrated in the schematic below.

[0238] In the preferred embodiments of this invention, the functionalgroup on this protein will be a thiol group and the chemically reactivegroup will be a maleimido-containing group such as MPA or GMBA(gamma-maleimide-butyralamide). The maleimido group is most selectivefor sulfhydryl groups on peptides when the pH of the reaction mixture iskept between 6.5 and 7.4. At pH 7.0, the rate of reaction of maleimidogroups with sulfhydryls is 1000-fold faster than with amines. A stablethioether linkage between the maleimido group and the sulfhydryl isformed which cannot be cleaved under physiological conditions, asdemonstrated in the following schematic.

[0239] A. Specific Labeling.

[0240] Preferably, the modified therapeutic agents of this invention aredesigned to specifically react with thiol groups on pulmonary proteinsor mobile blood proteins. Such reaction is preferably established bycovalent bonding of the therapeutic agent modified with a maleimide link(e.g. prepared from GMBS, MPA or other maleimides) to a thiol group on apulmonary protein, such as intra- or extra-cellular albumin, or a mobileblood protein such as serum albumin or IgG.

[0241] Under certain circumstances, specific labeling with maleimidesoffers several advantages over non-specific labeling of proteins withgroups such as NHS and sulfo-NHS. Thiol groups are less abundant in vivothan amino groups. Therefore, the maleimide-modified therapeutic agentsof this invention, i.e., maleimide therapeutic agents, will covalentlybond to fewer proteins. For example, in albumin (the most abundant bloodprotein) there is only a single thiol group. Thus, therapeuticagent-maleimide-albumin conjugates will tend to comprise approximately a1:1 molar ratio of therapeutic agent to albumin. In addition to albumin,IgG molecules (class II) also have free thiols. In the case of systemicdelivery, since IgG molecules and serum albumin make up the majority ofthe soluble protein in blood they also make up the majority of the freethiol groups in blood that are available to covalently bond tomaleimide-modified therapeutic agents.

[0242] Further, even among free thiol-containing blood proteins,including IgGs, specific labeling with maleimides leads to thepreferential formation of therapeutic agent-maleimide-albuminconjugates, due to the unique characteristics of albumin itself. Thesingle free thiol group of albumin, highly conserved among species, islocated at amino acid residue 34 (Cys³⁴). It has been demonstratedrecently that the Cys³⁴ of albumin has increased reactivity relative tofree thiols on other free thiol-containing proteins. This is due in partto the very low pK value of 5.5 for the Cys³⁴ of albumin. This is muchlower than typical pK values for cysteine residues in general, which aretypically about 8. Due to this low pK, under normal physiologicalconditions Cys³⁴ of albumin is predominantly in the ionized form, whichdramatically increases its reactivity. In addition to the low pK valueof Cys³⁴, another factor which enhances the reactivity of Cys³⁴ is itslocation, which is in a crevice close to the surface of one loop ofregion V of albumin. This location makes Cys³⁴ very available to ligandsof all kinds, and is an important factor in Cys³⁴'s biological role asfree radical trap and free thiol scavenger. These properties make Cys34highly reactive with maleimide-therapeutic agents, and the reaction rateacceleration can be as much as 1000-fold relative to rates of reactionof maleimide-therapeutic agents with other free-thiol containingproteins.

[0243] Another advantage of therapeutic agent-maleimide-albuminconjugates is the reproducibility associated with the 1:1 loading oftherapeutic agent to albumin specifically at Cys³⁴. Other techniques,such as glutaraldehyde, DCC, EDC and other chemical activations of, e.g,free amines, lack this selectivity. For example, albumin contains 52lysine residues, 25-30 of which are located on the surface of albuminand therefore accessible for conjugation. Activating these lysineresidues, or alternatively modifying therapeutic agents to couplethrough these lysine residues, results in a heterogenous population ofconjugates. Even if 1:1 molar ratios of therapeutic agent to albumin areemployed, the yield will consist of multiple conjugation products, somecontaining 0, 1, 2 or more therapeutic agents per albumin, and eachhaving therapeutic agents randomly coupled at any one or more of the25-30 available lysine sites. Given the numerous possible combinations,characterization of the exact composition and nature of each conjugatebatch becomes difficult, and batch-to-batch reproducibility is all butimpossible, making such conjugates less desirable as a therapeutic.Additionally, while it would seem that conjugation through lysineresidues of albumin would at least have the advantage of delivering moretherapeutic agent per albumin molecule, studies have shown that a 1:1ratio of therapeutic agent to albumin is preferred. In an article byStehle, et al., “The Loading Rate Determines Tumor Targeting propertiesof Methotrexate-Albumin Conjugates in Rats,” Anti-Cancer Drugs, Vol. 8,pp. 677-685 (1988), incorporated herein in its entirety, the authorsreport that a 1:1 ratio of the anti-cancer methotrexate to albuminconjugated via glutaraldehyde gave the most promising results. Theseconjugates were preferentially taken up by tumor cells, whereasconjugates bearing 5:1 to 20:1 methotrexate molecules had altered HPLCprofiles and were quickly taken up by the liver in vivo. It ispostulated that at these higher ratios, conformational changes toalbumin diminish its effectiveness as a therapeutic carrier.

[0244] Through controlled administration of maleimide-therapeutic agentsin vivo, one can control the specific labeling of albumin and IgG invivo. For systemic delivery via pulmonary administration, in typicaladministrations, 80-90% of the administered maleimide-therapeutic agentsthat reach the bloodstream will label albumin and less than 5% willlabel IgG. Trace labeling of free thiols such as glutathione will alsooccur. Such specific labeling is preferred for in vivo use as it permitsan accurate calculation of the estimated half-life of the administeredagent.

[0245] In addition to providing controlled specific in vivo labeling,maleimide-therapeutic agents can provide specific labeling of albumin orother proteins ex vivo. Such ex vivo labeling involves the addition ofmaleimide-therapeutic agents to a saline solution containing albumin orother protein. Once conjugation has occurred ex vivo with themaleimide-therapeutic agents, the saline solution can be administeredvia pulmonary delivery for in vivo treatment.

[0246] In contrast to NHS-therapeutic agents, maleimide-therapeuticagents are generally quite stable in the presence of aqueous solutionsand in the presence of free amines. Since maleimide-therapeutic agentswill only react with free thiols, protective groups are generally notnecessary to prevent the maleimide-therapeutic agents from reacting withitself. In addition, the increased stability of the modified therapeuticagent permits the use of further purification steps such as HPLC toprepare highly purified products suitable for in vivo use. Lastly, theincreased chemical stability provides a product with a longer shelflife.

[0247] B. Non-Specific Labeling.

[0248] The therapeutic agents of the invention may also be modified fornon-specific labeling of pulmonary or blood components. Bonds to aminogroups will also be employed, particularly with the formation of amidebonds for non-specific labeling. To form such bonds, one may use as achemically reactive group a wide variety of active carboxyl groups,particularly esters, where the hydroxyl moiety is physiologicallyacceptable at the levels required. While a number of different hydroxylgroups may be employed in these linking agents, the most convenientwould be N-hydroxysuccinimide (NHS) and N-hydroxy-sulfosuccinimide(sulfo-NHS).

[0249] Other linking agents which may be utilized are described in U.S.Pat. No. 5,612,034, which is hereby incorporated herein.

[0250] The various sites with which the chemically reactive group of themodified therapeutic agents may react in vivo include cells,particularly the alveolar cells and capillary endothelial cells thatmake up the alveoli in the lungs as well as red blood cells(erythrocytes) and platelets in the blood itself. The agents may alsoreact with pulmonary proteins, including membrane bound receptors,intra- and extra-cellular albumin, immunoglobulins, ferritin, andtransferrin, and serum proteins of the blood, such as immunoglobulins,including IgG and IgM, serum albumin, ferritin, steroid bindingproteins, transferrin, thyroxin binding protein, α-2-macroglobulin, andthe like. Those receptors with which the modified therapeutic agentsreact, which are not long-lived, will generally be eliminated from thehuman host within about three days. The proteins indicated above(including the proteins of the cells) will remain at least three days,and may remain five days or more (usually not exceeding 60 days, moreusually not exceeding 30 days) particularly as to the half life, basedon the concentration in the blood.

[0251] For the most part, for systemic delivery of the therapeuticagent, reaction will be with mobile components in the blood,particularly blood proteins and cells, more particularly blood proteinsand erythrocytes. By “mobile” is intended that the component does nothave a fixed situs for any extended period of time, generally notexceeding 5 minutes, more usually one minute, although some of the bloodcomponent may be relatively stationary for extended periods of time.Initially, there will be a relatively heterogeneous population offunctionalized proteins and cells. However, for the most part, thepopulation within a few days will vary substantially from the initialpopulation, depending upon the half-life of the functionalized proteinsin the blood stream. Therefore, usually within about three days or more,IgG will become the predominant functionalized protein in the bloodstream.

[0252] Usually, by day 5 post-administration, IgG, serum albumin anderythrocytes will be at least about 60 mole %, usually at least about 75mole %, of the conjugated components in blood, with IgG, IgM (to asubstantially lesser extent) and serum albumin being at least about 50mole %, usually at least about 75 mole %, more usually at least about 80mole %, of the non-cellular conjugated components.

[0253] The desired conjugates of non-specific modified therapeuticagents to blood components may be prepared in vivo by pulmonaryadministration of the modified therapeutic agents to the patient, whichmay be a human or other mammal. If desired, the subject conjugates mayalso be prepared ex vivo by combining a carrier protein or proteinsolution with modified therapeutic agents of the present invention,allowing covalent bonding of the modified therapeutic agents tofunctionalitieson the protein and then administering the conjugatedmixture to the host via pulmonary delivery.

[0254] 3. Diagnostic Agents and Modified Diagnostic Agents

[0255] Diagnostic agents are agents useful in imaging the mammalianvascular system and include such agents as position emission tomography(PET) agents, computerized tomography (CT) agents, magnetic resonanceimaging (MRI) agents, nuclear magnetic imaging agents (NMI), fluroscopyagents and ultrasound contrast agents.

[0256] The modified diagnostic agent of the present invention will, forthe most part, have the following formula: X-Y-Z.

[0257] In the formula, X is a diagnostic agent selected from PET agent,CT agents, MRI agents, NMI agents, fluroscopy agents and ultrasoundcontrast agents. Diagnostic agents of interest include radioisotopes ofsuch elements as iodine (I), including ¹²³I, ¹²⁵I, ¹³¹I, etc., barium(Ba), gadolinium (Gd), technetium (Tc), including ⁹⁹Tc, phosphorus (P),including ³¹P, iron (Fe), manganese (Mn), thallium (Tl), chromium (Cr),including ⁵¹Cr, carbon (C), including ¹⁴C, or the like, fluorescentlylabeled compounds, etc.

[0258] In the formula, Y is a linking group of from 0-30, more usuallyof from 2-12, preferably of from 4-12 atoms, particularly carbon,oxygen, phosphorous and nitrogen, more particularly carbon and oxygen,where the oxygen is preferably present as oxy ether, where Y may bealkylene, oxyalkylene, or polyoxyalkylene, where the oxyalkylene grouphas from 2-3 carbon atoms, and the like. A linking group of 0 atoms ispreferred when it is desired to place X as close to Z as possible.

[0259] In the formula, Z is a reactive entity, such as carboxy, carboxyester, where the ester group is of 1-8, more usually 1-6 carbon atoms,particularly a physiologically acceptable leaving group which activatesthe carboxy carbonyl for reaction with amino groups in an aqueoussystem, e.g. N-hydroxysuccinimide (NHS), N-hydroxysulfosuccinimide,(sulfo-NHS), maleimide, maleimide esters, maleimide acids,maleimide-benzoyl-succinimide (MBS), gamma-maleimido-butyryloxysuccinimide ester (GMBS) and maleimidopropionic acid (MPA),N-hydroxysuccinimide isocyanate, isothiocyanate, thiolester,thionocarboxylic acid ester, imino ester, mixed anhydride, e.g.carbodiimide anhydride, carbonate ester, etc. and the like. The reactiveentity Z will covalently bond to functionalities in vivo or ex vivo.

[0260] 4. Synthesis of Peptide Therapeutic Agents

[0261] A. Peptide Synthesis

[0262] Therapeutic agents according to the present invention that arepeptides may be synthesized by standard methods of solid phase peptidechemistry known to those of ordinary skill in the art. For example,peptides may be synthesized by solid phase chemistry techniquesfollowing the procedures described by Steward and Young (Steward, J. M.and Young, J. D., Solid Phase Peptide Synthesis, 2nd Ed., PierceChemical Company, Rockford, Ill., (1984) using an Applied Biosystemsynthesizer. Similarly, multiple peptide fragments may be synthesizedthen linked together to form larger peptides. These synthetic peptidescan also be made with amino acid substitutions at specific locations.

[0263] For solid phase peptide synthesis, a summary of the manytechniques may be found in J. M. Stewart and J. D. Young, Solid PhasePeptide Synthesis, W. H. Freeman Co. (San Francisco), 1963 and J.Meienhofer, Hormonal Proteins and Peptides, vol. 2, p. 46, AcademicPress (New York), 1973. For classical solution synthesis see G. Schroderand K. Lupke, The Peptides, Vol. 1, Acacemic Press (New York). Ingeneral, these methods comprise the sequential addition of one or moreamino acids or suitably protected amino acids to a growing peptidechain. Normally, either the amino or carboxyl group of the first aminoacid is protected by a suitable protecting group. The protected orderivatized amino acid is then either attached to an inert solid supportor utilized in solution by adding the next amino acid in the sequencehaving the complimentary (amino or carboxyl) group suitably protectedand under conditions suitable for forming the amide linkage. Theprotecting group is then removed from this newly added amino acidresidue and the next amino acid (suitably protected) is added, and theprocess is repeated.

[0264] After all the desired amino acids have been linked in the propersequence, any remaining protecting groups (and any solid support) areremoved sequentially or concurrently to afford the final polypeptide. Bysimple modification of this general procedure, it is possible to addmore than one amino acid at a time to a growing chain, for example, bycoupling (under conditions which do not racemize chiral centers) aprotected tripeptide with a properly protected dipeptide to form, afterdeprotection, a pentapeptide.

[0265] A particularly preferred method of preparing compounds of thepresent invention involves solid phase peptide synthesis wherein theamino acid α-N-terminal is protected by an acid or base sensitive group.Such protecting groups should hav the properties of being stable to theconditions of peptide linkage formation while being readily removablewithout destruction of the growing peptide chain or racemization of anyof the chiral centers contained therein. Suitable protecting groups are9-fluorenylmethyloxycarbonyl (Fmoc), t-butyloxycarbonyl (Boc),benzyloxycarbonyl (Cbz), biphenylisopropyloxycarbonyl ,t-amyloxycarbonyl, isobornyloxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, o-nitrophenylsulfenyl,2-cyano-t-butyloxycarbonyl, and the like. The9-fluorenyl-methyloxycarbonyl (Fmoc) protecting group is particularlypreferred for the synthesis of the peptides of the present invention.Other preferred side chain protecting groups are, for side chain aminogroups like lysine and arginine, 2,2,5,7,8-pentamethylchroman-6-sulfonyl(pmc), nitro, p-toluenesulfonyl, 4-methoxybenzene-sulfonyl, Cbz, Boc,and adamantyloxycarbonyl; for tyrosine, benzyl,o-bromobenzyloxycarbonyl, 2,6-dichlorobenzyl, isopropyl, t-butyl (t-Bu),cyclohexyl, cyclopenyl and acetyl (Ac); for serine, t-butyl, benzyl andtetrahydropyranyl; for histidine, trityl, benzyl, Cbz, p-toluenesulfonyland 2,4-dinitrophenyl; for tryptophan, formyl; for asparticacid andglutamic acid, benzyl and t-butyl and for cysteine, triphenylmethyl(trityl).

[0266] In the solid phase peptide synthesis method, the α-C-terminalamino acid is attached to a suitable solid support or resin. Suitablesolid supports useful for the above synthesis are those materials whichare inert to the reagents and reaction conditions of the stepwisecondensation-deprotection reactions, as well as being insoluble in themedia used. The preferred solid support for synthesis of α-C-terminalcarboxy peptides is 4-hydroxymethylphenoxymethyl-copoly(styrene-1%divinylbenzene). The preferred solid support for α-C-terminal amidepeptides is the4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxyacetamidoethyl resinavailable from Applied Biosystems (Foster City, Calif.). Theα-C-terminal amino acid is coupled to the resin by means ofN,N′-dicyclohexylcarbodiimide (DCC), N,N′-diisopropylcarbodiimide (DIC)or O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium-hexafluorophosphate(HBTU), with or without 4-dimethylaminopyridine (DMAP),1-hydroxybenzotriazole (HOBT),benzotriazol-1-yloxy-tris(dimethylamino)phosphonium-hexafluorophosphate(BOP) or bis(2-oxo-3-oxazolidinyl)phosphine chloride (BOPCl), mediatedcoupling for from about 1 to about 24 hours at a temperature of between10° and 50° C. in a solvent such as dichloromethane or DMF.

[0267] When the solid support is4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resin,the Fmoc group is cleaved with a secondary amine, preferably piperidine,prior to coupling with the α-C-terminal amino acid as described above.The preferred method for coupling to the deprotected4-(2′,4′-dimethoxyphenyl-Fmoc-aminomethyl)phenoxy-acetamidoethyl resinis O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluoro-phosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.) in DMF. Thecoupling of successive protected amino acids can be carried out in anautomatic polypeptide synthesizer as is well known in the art. In apreferred embodiment, the α-N-terminal amino acids of the growingpeptide chain are protected with Fmoc. The removal of the Fmocprotecting group from the α-N-terminal side of the growing peptide isaccomplished by treatment with a secondary amine, preferably piperidine.Each protected amino acid is then introduced in about 3-fold molarexcess, and the coupling is preferably carried out in DMF. The couplingagent is normallyO-benzotriazol-1-yl-N,N,N′,N′-tetramethyluroniumhexafluorophosphate(HBTU, 1 equiv.) and 1-hydroxybenzotriazole (HOBT, 1 equiv.).

[0268] At the end of the solid phase synthesis, the polypeptide isremoved from the resin and deprotected, either in successively or in asingle operation. Removal of the polypeptide and deprotection can beaccomplished in a single operation by treating the resin-boundpolypeptide with a cleavage reagent comprising thioanisole, water,ethanedithiol and trifluoroacetic acid. In cases wherein theα-C-terminal of the polypeptide is an alkylamide, the resin is cleavedby aminolysis with an alkylamine. Alternatively, the peptide may beremoved by transesterification, e.g. with methanol, followed byaminolysis or by direct transamidation. The protected peptide may bepurified at this point or taken to the next step directly. The removalof the side chain protecting groups is accomplished using the cleavagecocktail described above. The fully deprotected peptide is purified by asequence of chromatographic steps employing any or all of the followingtypes: ion exchange on a weakly basic resin (acetate form); hydrophobicadsorption chromatography on underivitized polystyrene-divinylbenzene(for example, Amberlite XAD); silica gel adsorption chromatography; ionexchange chromatography on carboxymethylcellulose; partitionchromatography, e.g. on Sephadex G-25, LH-20 or countercurrentdistribution; high performance liquid chromatography (HPLC), especiallyreverse-phase HPLC on octyl- or octadecylsilyl-silica bonded phasecolumn packing.

[0269] Molecular weights of these ITPs are determined using Fast AtomBombardment (FAB) Mass Spectroscopy.

[0270] (1) N-Terminal Protective Groups

[0271] As discussed above, the term “N-protecting group” refers to thosegroups intended to protect the α-N-terminal of an amino acid or peptideor to otherwise protect the amino group of an amino acid or peptideagainst undesirable reactions during synthetic procedures. Commonly usedN-protecting groups are disclosed in Greene, “Protective Groups InOrganic Synthesis,” (John Wiley & Sons, New York (1981)), which ishereby incorporated by reference. Additionally, protecting groups can beused as pro-drugs which are readily cleaved in vivo, for example, byenzymatic hydrolysis, to release the biologically active parent.α-N-protecting groups comprise loweralkanoyl groups such as formyl,acetyl (“Ac”), propionyl, pivaloyl, t-butylacetyl and the like; otheracyl groups include 2-chloroacetyl, 2-bromoacetyl, trifluoroacetyl,trichloroacetyl, phthalyl, o-nitrophenoxyacetyl, -chlorobutyryl,benzoyl, 4-chlorobenzoyl, 4-bromobenzoyl, 4-nitrobenzoyl and the like;sulfonyl groups such as benzenesulfonyl, p-toluenesulfonyl and the like;carbamate forming groups such as benzyloxycarbonyl,p-chlorobenzyloxycarbonyl, p-methoxybenzyloxycarbonyl,p-nitrobenzyloxycarbonyl, 2-nitrobenzyloxycarbonyl,p-bromobenzyloxycarbonyl, 3,4-dimethoxybenzyloxycarbonyl,3,5-dimethoxybenzyloxycarbonyl, 2,4-dimethoxybenzyloxycarbonyl,4-ethoxybenzyloxycarbonyl, 2-nitro-4,5-dimethoxybenzyloxycarbonyl,3,4,5-trimethoxybenzyloxycarbonyl,1-(p-biphenylyl)-1-methylethoxycarbonyl,α,α-dimethyl-3,5-dimethoxybenzyloxycarbonyl, benzhydryloxycarbonyl,t-butyloxycarbonyl (Boc), diisopropylmethoxycarbonyl,isopropyloxycarbonyl, ethoxycarbonyl, methoxycarbonyl, allyloxycarbonyl,2,2,2,-trichloroethoxycarbonyl, phenoxycarbonyl, 4-nitrophenoxycarbonyl,fluorenyl-9-methoxycarbonyl, cyclopentyloxycarbonyl,adamantyloxycarbonyl, cyclohexyloxycarbonyl, phenylthiocarbonyl and thelike; arylalkyl groups such as benzyl, triphenylmethyl, benzyloxymethyl,9-fluorenylmethyloxycarbonyl (Fmoc) and the like and silyl groups suchas trimethylsilyl and the like.

[0272] (2) Carboxy Protective Groups

[0273] As discussed above, the term “carboxy protecting group” refers toa carboxylic acid protecting ester or amide group employed to block orprotect the carboxylic acid functionality while the reactions involvingother functional sites of the compound are performed. Carboxy protectinggroups are disclosed in Greene, “Protective Groups in Organic Synthesis”pp. 152-186 (1981), which is hereby incorporated by reference.Additionally, a carboxy protecting group can be used as a pro-drugwhereby the carboxy protecting group can be readily cleaved in vivo, forexample by enzymatic hydrolysis, to release the biologically activeparent. Such carboxy protecting groups are well known to those skilledin the art, having been extensively used in the protection of carboxylgroups in the penicillin and cephalosporin fields as described in U.S.Pat. Nos. 3,840,556 and 3,719,667, the disclosures of which are herebyincorporated herein by reference. Representative carboxy protectinggroups are C₁-C₈ loweralkyl (e.g., methyl, ethyl or t-butyl and thelike); arylalkyl such as phenethyl or benzyl and substituted derivativesthereof such as alkoxybenzyl or nitrobenzyl groups and the like;arylalkenyl such as phenylethenyl and the like; aryl and substitutedderivatives thereof such as 5-indanyl and the like; dialkylaminoalkylsuch as dimethylaminoethyl and the like); alkanoyloxyalkyl groups suchas acetoxymethyl, butyryloxymethyl, valeryloxymethyl,isobutyryloxymethyl, isovaleryloxymethyl, 1-(propionyloxy)-1-ethyl,1-(pivaloyloxyl)-1-ethyl, 1-methyl-1-(propionyloxy)-1-ethyl,pivaloyloxymethyl, propionyloxymethyl and the like;cycloalkanoyloxyalkyl groups such as cyclopropylcarbonyloxymethyl,cyclobutylcarbonyloxymethyl, cyclopentylcarbonyloxymethyl,cyclohexylcarbonyloxymethyl and the like; aroyloxyalkyl such asbenzoyloxymethyl, benzoyloxyethyl and the like;arylalkylcarbonyloxyalkyl such as benzylcarbonyloxymethyl,2-benzylcarbonyloxyethyl and the like; alkoxycarbonylalkyl orcycloalkyloxycarbonylalkyl such as methoxycarbonylmethyl,cyclohexyloxycarbonylmethyl, 1-methoxycarbonyl-1-ethyl and the like;alkoxycarbonyloxyalkyl or cycloalkyloxycarbonyloxyalkyl such asmethoxycarbonyloxymethyl, t-butyloxycarbonyloxymethyl,1-ethoxycarbonyloxy-1-ethyl, 1-cyclohexyloxycarbonyloxy-1-ethyl and thelike; aryloxycarbonyloxyalkyl such as 2-(phenoxycarbonyloxy)ethyl,2-(5-indanyloxycarbonyloxy)ethyl and the like;alkoxyalkylcarbonyloxyalkyl such as2-(1-methoxy-2-methylpropan-2-oyloxy)ethyl and like;arylalkyloxycarbonyloxyalkyl such as 2-(benzyloxycarbonyloxy)ethyl andthe like; arylalkenyloxycarbonyloxyalkyl such as2-(3-phenylpropen-2-yloxycarbonyloxy)ethyl and the like;alkoxycarbonylaminoalkyl such as t-butyloxycarbonylaminomethyl and thelike; alkylaminocarbonylaminoalkyl such asmethylaminocarbonylaminomethyl and the like; alkanoylaminoalkyl such asacetylaminomethyl and the like; heterocycliccarbonyloxyalkyl such as4-methylpiperazinylcarbonyloxymethyl and the like;dialkylaminocarbonylalkyl such as dimethylaminocarbonylmethyl,diethylaminocarbonylmethyl and the like;(5-(loweralkyl)-2-oxo-1,3-dioxolen-4-yl)alkyl such as(5-t-butyl-2-oxo-1,3-dioxolen4-yl)methyl and the like; and(5-phenyl-2-oxo-1,3-dioxolen4-yl)alkyl such as(5-phenyl-2-oxo-1,3-dioxolen4-yl)methyl and the like.

[0274] Representative amide carboxy protecting groups are aminocarbonyland loweralkylaminocarbonyl groups.

[0275] Preferred carboxy-protected compounds of the invention arecompounds wherein the protected carboxy group is a loweralkyl,cycloalkyl or arylalkyl ester, for example, methyl ester, ethyl ester,propyl ester, isopropyl ester, butyl ester, sec-butyl ester, isobutylester, amyl ester, isoamyl ester, octyl ester, cyclohexyl ester,phenylethyl ester and the like or an alkanoyloxyalkyl,cycloalkanoyloxyalkyl, aroyloxyalkyl or an arylalkylcarbonyloxyalkylester. Preferred amide carboxy protecting groups areloweralkylaminocarbonyl groups. For example, aspartic acid may beprotected at the α-C-terminal by an acid labile group (e.g. t-butyl) andprotected at the β-C-terminal by a hydrogenation labile group (e.g.benzyl) then deprotected selectively during synthesis.

[0276] B. Peptide Modification

[0277] The manner of producing the modified peptides of the presentinvention will vary widely, depending upon the nature of the variouselements comprising the peptide. The synthetic procedures will beselected so as to be simple, provide for high yields, and allow for ahighly purified stable product. Normally, the chemically reactive groupwill be created at the last stage of the synthesis, for example, with acarboxyl group, esterification to form an active ester. Specific methodsfor the production of modified peptides of the present invention aredescribed below.

[0278] Specifically, the selected peptide is modified with the linkinggroup only at either the N-terminus, C-terminus or interior of thepeptide. The therapeutic activity of this modified peptide-linking groupis then assayed. If the therapeutic activity is not reduced dramatically(i.e., reduced less than 10-fold), then the stability of the modifiedpeptide-linking group is measured by its in vivo lifetime. If thestability is not improved to a desired level, then the peptide ismodified at an alternative site, and the procedure is repeated until adesired level of therapeutic and stability is achieved.

[0279] More specifically, each peptide selected to undergo modificationwith a linking group and a reactive group will be modified according tothe following criteria: if a terminal carboxylic group is available onthe peptide and is not critical for the retention of therapeuticactivity, and no other sensitive functional group is present on thepeptide, then the carboxylic acid will be chosen as attachment point forthe linking group-reactive group modification. If the terminalcarboxylic group is involved in therapeutic activity, or if nocarboxylic acids are available, then any other sensitive functionalgroup not critical for the retention of therapeutic activity will beselected as the attachment point for the linking group-reactive entitymodification. If several sensetive functional groups are available on aa peptide, a combination of protecting groups will be used in such a waythat after addition of the linking group/reactive entity anddeprotection of all the protected sensetive functional groups, retentionof therapeutic activity is still obtained. If no sensetive functionalgroups are available on the peptide, or if a simpler modification routeis desired, synthetic efforts will allow for a modification of theoriginal peptide in such a way that retention of therapeutic ismaintained. In this case the modification will occur at the opposite endof the peptide

[0280] An NHS derivative may be synthesized from a carboxylic acid inabsence of other sensetive functional groups in the peptide.Specifically, such a peptide is reacted with N-hydroxysuccinimide inanhydrous CH₂Cl₂ and EDC, and the product is purified by chromatographyor recrystallized from the appropriate solvent system to give the NHSderivative.

[0281] Alternatively, an NHS derivative may be synthesized from apeptide that contains an amino and/or thiol group and a carboxylic acid.When a free amino or thiol group is present in the molecule, it ispreferable to protect these sensetive functional groups prior to performthe addition of the NHS derivative. For instance, if the moleculecontains a free amino group, a transformation of the amine into a Fmocor preferably into a tBoc protected amine is necessary prior to performthe chemistry described above. The amine functionality will not bedeprotected after preparation of the NHS derivative. Therefore thismethod applies only to a compound whose amine group is not required tobe freed to induce the desired therapeutic effect. If the amino groupneeds to be freed to retain the original properties of the molecule,then another type of chemistry described below has to be performed.

[0282] In addition, an NHS derivative may be synthesized from a peptidecontaining an amino or a thiol group and no carboxylic acid. When theselected molecule contains no carboxylic acid, an array of bifunctionallinking groups can be used to convert the molecule into a reactive NHSderivative. For instance, ethylene glycol-bis(succinimydylsuccinate)(EGS) and triethylamine dissolved in DMF and added to the free aminocontaining molecule (with a ratio of 10:1 in favor of EGS) will producethe mono NHS derivative. To produce an NHS derivative from a thiolderivatized molecule, one can use N-[-maleimidobutyryloxy]succinimideester (GMBS) and triethylamine in DMF. The maleimido group will reactwith the free thiol and the NHS derivative will be purified from thereaction mixture by chromatography on silica or by HPLC.

[0283] An NHS derivative may also be synthesized from a peptidecontaining multiple sensetive functional groups. Each case will have tobe analyzed and solved in a different manner. However, thanks to thelarge array of protecting groups and bifunctional linking groups thatare commercially available, this invention is applicable to any peptidewith preferably one chemical step only to modify the peptide (asdescribed above) or two steps (as described above involving priorprotection of a sensitive group) or three steps (protection, activationand deprotection). Under exceptional circumstances only, would multiplesteps (beyond three steps) synthesis be required to transform a peptideinto an active NHS or maleimide derivative.

[0284] A maleimide derivative may also be synthesized from a peptidecontaining a free amino group and a free carboxylic acid. To produce amaleimide derivative from a amino derivatized molecule, one can useN-[γ-maleimidobutyryloxy]succinimide ester (GMBS) and triethylamine inDMF. The succinimide ester group will react with the free amino and themaleimide derivative will be purified from the reaction mixture bycrystallization or by chromatography on silica or by HPLC.

[0285] Finally, a maleimide derivative may be synthesized from a peptidecontaining multiple other sensetive functional groups and no freecarboxylic acids. When the selected molecule contains no carboxylicacid, an array of bifunctional crosslinking reagents can be used toconvert the molecule into a reactive NHS derivative. For instancemaleimidopropionic acid (MPA) can be coupled to the free amine toproduce a maleimide derivative through reaction of the free amine withthe carboxylic group of MPA using HBTU/HOBt/DIEA activation in DMF.

[0286] Many other commercially available heterobifunctional crosslinkingreagents can alternatively be used when needed. A large number ofbifunctional compounds are available for linking to entities.Illustrative reagents include: azidobenzoyl hydrazide,N-[4-(p-azidosalicylamino)butyl]-3′-[2′-pyridyldithio)propionamide),bis-sulfosuccinimidyl suberate, dimethyl adipimidate, disuccinimidyltartrate, N-y-maleimidobutyryloxysuccinimide ester, N-hydroxysulfosuccinimidyl-4-azidobenzoate, N-succinimidyl[4-azidophenyl]-1,3′-dithiopropionate, N-succinimidyl[4-iodoacetyl]aminobenzoate, glutaraldehyde, and succinimidyl4-[N-maleimidomethyl]cyclohexane-1-carboxylate.

[0287] 5. Synthesis of Modified Organic Therapeutic Agents

[0288] Similar to procedures for modified peptide therapeutics, thesynthetic procedures used to prepare modified organic therapeutics willalso be selected so as to be simple, provide for high yields, and allowfor a highly purified product. Normally, the chemically reactive groupwill be created as the last stage, for example, with a carboxyl group,esterification to form an active ester will be the last step of thesynthesis. Methods for the production and/or modification of organictherapeutic agents of the present invention are described in theExamples below.

[0289] Each organic therapeutic agent selected to undergo thederivatization with a linking group and a reactive group will bemodified according to the following criteria: Generally, the therapeuticagents are commercially available. If not, they can be synthesized byprocedures well known in the art. As a first step, the therapeuticallyactive region of the therapeutic agent is identified. Next, thetherapeutic agent is modified at a site sufficiently far away from theactive portion to prevent a potential interference between the modifieddrug and the target site of the drug, such that the modified agentsubstantially retains its therapeutic activity, (i.e. the therapeuticactivity is reduced by no more than 10 fold). Finally, keeping constantthe site of chemical modification, optimize the biological activity ofthe modified agent by modifying the length and nature of the linkinggroup.

[0290] If a carboxylic group, not critical for the retention ofpharmacological activity is available on the original molecule and noother reactive functionality is present on the molecule, then thecarboxylic acid will be chosen as attachment point for the linkinggroup-reactive entity modification. If no carboxylic acids areavailable, then any other functionalities not critical for the retentionof pharmacological activity will be selected as attachment point for thelinking group-reactive entity modification. If several functionalitiesare available on a therapeutic agent, a combination of protecting groupswill be used in such a way that after addition of the linkinggroup/reactive entity and deprotection of all the protectedfunctionalities, retention of pharmacological activity is stillobtained. If no functionalities are available on the therapeutic agent,synthetic efforts will allow for a modification of the original parentdrug in such a way that retention of biological activity and retentionof receptor or target specificity is obtained.

[0291] Where the derivatized therapeutic agent of the present inventionrepresents a derivatized enzyme inhibitor will generally havesubstantially lower IC₅₀'s generally in the range of about 0.5-0.01 ofthe IC₅₀ of the parent molecule. Desirably, the IC₅₀ should be not lessthan 0.05, preferably not less than about 0.1. In view of the varyingIC₅₀'s, the amount of the derivatized therapeutic agent administeredwill also vary.

[0292] The determination of the nature and length of the linking groupwill be performed through an empirical optimization phase and will bemeasured by the retention or the loss of biological activity. Forinstance, with a given inhibitor enzyme interactions, an iteration ofthe modification of the nature and the length of the linking group and ameasure of the biological enzymatic activity may be necessary todetermine the most favored linking group length and nature. Preferably ashort hydrophilic 4-12 atom linking group easily synthesized will befavored to start the iteration process.

[0293] In the case of radiolabeled therapeutic agents, a minimumdistance from the target has to be respected based on the nature of theisotope and its penetration. The length and nature of the linking groupsare not as important as they are for an enzyme inhibitor combination.For instance an isotope that emits a beta rays like ³²P should bepositioned within 5 mm from the target to have maximum efficiency (99%)with limited or no effect coming from a small change on the nature andlength of the linking group.

[0294] 6. Synthesis of Modified Diagnostic Agents

[0295] The manner of producing the modified diagnostic agents of thepresent invention will vary widely, depending upon the nature of thevarious elements comprising the molecule. The synthetic procedures willbe selected so as to be simple, provide for high yields, and allow for ahighly purified product. Normally, the chemically reactive group will becreated as the last stage, for example, with a carboxyl group,esterification to form an active ester will be the last step of thesynthesis. Methods for the production of the diagnostic agents of thepresent invention are described in the Examples.

[0296] 7. Pulmonary Formulations and Delivery Methods

[0297] A further aspect of the invention is directed to aerosolcompositions for treatment of the symptoms of pulmonary conditions. Withtherapeutic agents capable of forming a covalent bond with a pulmonaryfluid protein. Alternatively, the therapeutic agent may be firstconjugated with the pulmonary solution protein prior to administration.

[0298] The aerosol compositions may be composed of an aqueous solutionsuitable for inhalation consisting of at least 2.5% by weight (morepreferably between about 3% and 10% by weight, and most preferably atleast about 5% by weight) of a derivatized therapeutic agent. Thedroplets of the aerosol should be 10μ or less in diameter to maximizedeposition in the lung alveoli rather than the throat or upperrespiratory tract.

[0299] The invention also features inhaler devices for administration ofthe inhalable compositions (or medicaments) of the subject invention. Inone aspect of the invention, the inhaler device comprises a housingdefining a chamber which contains a dry powder. The dry powder iscomposed of therapeutic agent compound present in an amount that, uponadministration will bind to pulmonary proteins. Alternatively, the agentmay be covalently bonded to pulmonary proteins ex vivo prior toadministration. At least 50% (preferably at least 70%, and morepreferably at least 90%) of the powder consists of primary particleswhich have a diameter of 10 μm or less, and which may be agglomeratedinto larger particles or agglomerates which readily break down intoprimary particles upon inhalation from the device. The chamber has anopening through which the medicament can be drawn by inhalation by apatient.

[0300] In another aspect of the invention, the inhaler device comprisesa vessel containing an inhalable medicament in the form of an aqueoussolution suspended in a compressed or liquified propellant gas. At least2.5% by weight (preferably at least 3%, more preferably at least 4%,even more preferably at least 5% and most preferably between 6 and 10%)of the aqueous solution is a pH-raising buffer compound.

[0301] The inhaler device may also have a housing defining a port ontowhich the vessel is mounted, a lumen in communication with the port, anda mechanism for controllably releasing the propellant from the vesselinto the lumen, thereby releasing the suspended medicament from thevessel into the lumen. The lumen is configured to route the medicamentsuspended in the propellant into the respiratory system of the patient.

[0302] 8. Therapeutic Uses of Modified Therapeutic Agents

[0303] The modified therapeutic agents of the invention find numeroususes, as enumerated below.

[0304] A. Therapeutic Uses of Modified Antineoplastic Agent

[0305] The antineoplastic agents of the invention, including but notlimited to those specified in the examples, possess anti-angiogenicactivity. As modified antineoplastic agents having anti-angiogenicactivity, the compounds of the present invention are useful in thetreatment of a variety of diseases, for example primary and metastaticsolid tumors and carcinomas of the breast; colon; rectum; lung;oropharynx; hypopharynx; esophagus; stomach. Pancreas; liver;gallbladder; bile ducts; small intestine; urinary tract includingkidney, bladder and urothelium; female genital tract including cervix,uterus, endometrium, ovaries, choriocarcinoma and festationaltrophoblastic disease; male genital tract including prostate, seminalvesicles, testes and germ cells tumors; endocrine glands includingthyroid, adrenal and pituitary; skin including hmangiomas, melanomas,sarcomas arising from bone of soft tissues and Karposi's sarcoma, Wilm'stumor, rhabdomyosarcoma; tumor of the head and neck, brain, nerves,eyes, and meninges including astrocytomas, gliomas, glioblastomas,retinoblastomas, neuromas, neuroblastomas; tumors of the bone marrow andhematopoeitic tumors, solid tumors arising from hematopoieticmalignancies such as leukemias and including chloromas, plasmocytomas,plagues and tumors of mycosis fungoides and cutaneous T-celllymphoma/leukemia; acute lymphotic, actute granulocytic and chronicgranulocytic leukemia; lymphomas including both Hodgkin's andnon-Hodgkin's lymphomas; prophylaxis of autoimmune diseases includingrheumatoid, immune and degenerative arthritis; ocular diseases includingdiabethic retinopathy; retinopathy of prematurity; corneal graftrejection, retrolental fibroplasia, neovascular glaucoma, rubeosis,retinal neovascularization due to macular degeneration and hypoxia;abnormal neovascularization conditions of the eye; skin diseasesincluding psoriasis; blood vessel diseases including hemagiomas andcapillary proliferatrion withinatherosclerotic plaques; myocardialangiogenesis; plaque neovascularization; hemophiliac joints;angiofibroma; wound granulation; dieases charadterized by excessive orabnormal stimulation of endothelial cells including intestinal adhesion,Crohn's disease, atheroscelrosis, scleroderma and hypertrophic scars anddiseases which have angiogenesis as a pathological consequence includingulcers (Helicobacter pilori); rheumatoid arthritis, osteogenic sarcoma,osteoarthritis, osteopenias such as osteoporosis, periodontitis,gingivitis, corneal epidermal or gastric ulceration, and tumormetastasis, invasion and growth, retinopathies, wound healing (ocularinflammation, soft and osseous tissue disease, gingivitis/periodontaldisease), vascular disease (restenosis) annuerysm inflammation,autoimmune diseases, and rare cancers such as choriocarcinoma.

[0306] The compounds of the present invention may also be useful for theprevention of metastases from the tumors described above either whenused alone or in combination with radiotherapy and/or otherchemotherapeutic treatments conventionally administered to patients fortreating angiogenic diseases. For example, when used in the treatment ofsolid tumors, compounds of the present invention may be administeredwith chemotherapeutic agents such as alpha-inferon, COMP(cyclophosphamnide, vincristine, methotraxate and prednisone),etoposide, mBACOD (methotraxate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PROMACE/MOPP(prednisone, methotrexate, doxirubicin, cyclophaophamide, taxol,etoposide/mechloetamine, vincristine, prednisone and procarbazine),vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate,platelet factor-4, angiostatin, LM-609, SU-101, CM-101, techgalan,thalidomide, SP-PG and the like. Other chemotherapeutic agents includealkylating agents such as nitrogen mustards including mechloethamine,melphanchloambucil, cyclophaosphamide, and ifosfamide; nirrosdoureasincluding carmustine, lomustine, semustine and streptozocin; alkylsulfonates icluding busulfan; triazines including dacarbazine;ethyenimines including thiotepa na dhexamethylmelanine; folic acidanalogs including methotraxate; pyrimidine analogs including 5-FU,cytosine arabinoside; purine analogs including 6-mercaptopurine and6-thioguanine; antitumor antibiotics including actinomycin D; theanthraqcyclines including doxorubicin, bleomycin, mitomycin C andmethramycin; hormones and hormones antagonists including tamoxifen andcorticosteroids and mioscellaneous agnets including cisplatin andbrequinar; fragments of plasminogen (kringle-5) as well as fragmentsfrom other integrin-binding substrates. For insnance, a tumor may betreated conventionally with surgery, radiation or chemiotherapy andadministration of modified antineoplastic agents to extend the dormancyof micrometastasis and to inhibit the growth of any residual primarytumor.

[0307] B. Therapeutic Uses of Modified Matrix Metalloprotease Inhibitors

[0308] The MMPIs of the invention, including but not limited to thosespecified in the examples, possess anti-angiogenic activity. As modifiedmatrix metalloprotease inhibitors having anti-angiogenic activity, thecompounds of the present invention are useful in the treatment of avariety of diseases, for example primary and metastatic solid tumors andcarcinomas of the breast; colon; rectum; lung; oropharynx; hypopharynx;esophagus; stomach. Pancreas; liver; gallbladder; bile ducts; smallintestine; urinary tract including kidney, bladder and urothelium;female genital tract including cervix, uterus, ovaries, choriocarcinomaand festational trophoblastic disease; male genital tract includingprostate, seminal vesicles, testes and germ cells tumors; endocrineglands including thyroid, adrenal and pituitary; skin includinghmangiomas, melanomas, sarcomas arising from bone of soft tissues andKarposi's sarcoma; tumor of the nrain, nerves, eyes, and meningesincluding astrocytomas, gliomas, glioblastomas, retinoblastomas,neuromas, neuroblastomas; tumors of the bone marrow and hematopoeitictumors, solid tumors arising from hematopoietic malignancies such asleukemias and including chloromas, plasmocytomas, plagues and tumors ofmycosis fungoides and cutaneous T-cell lymphoma/leukemia; lymphomasincluding both Hodgkin's and non-Hodgkin's lymphomas; prophylaxis ofautoimmune diseases including rheumatoid, immune and degenerativearthritis; ocular diseases including diabethic retinopathy; retinopathyof prematurity; corneal graft rejection, retrolental fibroplasia,neovascular glaucoma, rubeosis, retinal neovascularization due tomacular degeneration and hypoxia; abnormal neovascularization conditionsof the eye; skin diseases including psoriasis; blood vessel diseasesincluding hemagiomas and capillary proliferatrion withinatheroscleroticplaques; myocardial angiogenesis; plaque neovascularization; hemophiliacjoints; angiofibroma; wound granulation; dieases charadterized byexcessive or abnormal stimulation of endothelial cells includingintestinal adhesion, Crohn's disease, atheroscelrosis, scleroderma andhypertrophic scars and diseases which have angiogenesis as apathological consequence including ulcers (Helicobacter pilori);rheumatoid arthritis, osteoarthritis, osteopenias such as osteoporosis,periodontitis, gingivitis, corneal epidermal or gastric ulceration, andtumor metastasis, invasion and growth, retinopathies, wound healing(ocular inflammation, soft and osseous tissue disease,gingivitis/periodontal disease), vascular disease (restenosis) annuerysminflammation and and autoimmune diseases. Another use is as birthcontrol agent which inhibits ovulation and establishment of theplacenta.

[0309] The compounds of the present invention may also be useful for theprevention of metastases from the tumors described above either whenused alone or in combination with radiotherapy and/or otherchemotherapeutic treatments conventionally administered to patients fortreating angiogenic diseases. For example, when used in the treatment ofsolid tumors, compounds of the present invention may be administeredwith chemotherapeutic agents such as alpha-inferon, COMP(cyclophosphamnide, vincristine, methotraxate and prednisone),etoposide, mBACOD (methotraxate, bleomycin, doxorubicin,cyclophosphamide, vincristine and dexamethasone), PROMACE/MOPP(prednisone, methotrexate, doxirubicin, cyclophaophamide, taxol,etoposide/mechloetamine, vincristine, prednisone and procarbazine),vincristine, vinblastine, angioinhibins, TNP-470, pentosan polysulfate,platelet factor-4, angiostatin, LM-609, SU-101, CM-101, techgalan,thalidomide, SP-PG and the like. Other chemotherapeutic agents includealkylating agents such as nitrogen mustards including mechloethamine,melphanchloambucil, cyclophaosphamide, and ifosfamide; nirrosdoureasincluding carmustine, lomustine, semustine and streptozocin; alkylsulfonates icluding busulfan; triazines including dacarbazine;ethyenimines including thiotepa na dhexamethylmelanine; folic acidanalogs including methotraxate; pyrimidine analogs including 5-FU,cytosine arabinoside; purine analogs including 6-mercaptopurine and6-thioguanine; antitumor antibiotics including actinomycin D; theanthraqcyclines including doxorubicin, bleomycin, mitomycin C andmethramycin; hormones and hormones antagonists including tamoxifen andcorticosteroids and mioscellaneous agnets including cisplatin andbrequinar; fragments of plasminogen (kringle-5) as well as fragmentsfrom other integrin-binding substrates. For instance, a tumor may betreated conventionally with surgery, radiation or chemiotherapy and themodified MMPI molecules of the invention to extend the dormancy ofmicrometastasis and to inhibit the growth of any residual primary tumor.

[0310] It has also been found that hydroxamic acid MMPIs can inhibit theproduction of the cytokine tumor necrosis factor (TNF) (Mohler et al.,Nature, 1994, 370, 218-220; Gearing AJH et al., Nature 1994, 370,555-557; McGeehan G M et al., Nature 1994, 370, 558-561). Compoundswhich inhibit the production or action of TNF are thought to bepotentially useful for the treatment or prophylaxis of manyinflammatory, infectious, immunological or malignant diseases. Theseinclude, but are not restricted to, septic shock, haemodynamic shock andsepsis syndrome, post ischaemic reperfusion injury, malaria, Crohn'sdisease, mycobacterial infection, meningitis, psoriasis, congestiveheart failure, fibrotic disease, cachexia, graft rejection, cancer,autoimmune disease, rheumatic arthritis, multiple scleroris, radationdamage, toxicity following administration of immunosuppressivemonoclonal antibodies such as OKT3 or CAMPATH-1 and hyperoxic alveolarinjury. Since excessive TNF production has been noted in severaldiseases or conditions also characterized by MMP-mediated tissuedegradation, compounds which inhibit both MMPs and TNF production mayhave particular advantages in the treatment or prophylaxis of diseasesor conditions in which both mechanisms are involved.

[0311] The compounds of the present invention inhibit various enzymesfrom the matrix metalloproteinase family such as collagenase, whichinitiates collagen breakdown, stromelysin (protoglycanase), andgelatinase, and hence are useful for the treatment of matrix metalloendoproteinase diseases. There is evidence implicating collagenase asone of the key enzymes in the breakdown of articular cartilage and bonein rheumatoid arthritis (Arthritis and Rheumatism, 20, 1231-1239, 1977).Potent inhibitors of collagenase and other metalloproteases involved intissue degradation are useful in the treatment of rheumatoid arthritisand related diseases in which collagenolytic activity is important.Inhibitors of metalloproteases of this type can therefore be used intreating or preventing conditions which involve tissue breakdown; theyare therefore useful in the treatment of arthropathy, dermatologicalconditions, bone resorption, inflammatory diseases and tumour invasionand in the promotion of wound healing. Specifically, compounds of thepresent invention may be useful in the treatment of osteopenias such asosteoporosis, rheumatoid arthritis, osteoarthritis, periodontitis,gingivitis, corneal ulceration and tumour invasion.

[0312] C. Therapeutic Uses of Oxytocin

[0313] A conjugated oxytocin may be used to aid lactation and help relaxthe pelvis prior to birth. It could also be used to prevent post partumuterine hemorrage.

[0314] D. Therapeutic Uses of Cholecystokinin (CCK)

[0315] A conjugated CCK could be used in diagnostic studies of the gallbladder or in chronic cholecystisis.

[0316] E. Therapeutic Uses of Antihypertensive Agents

[0317] Antihypertensive agents are used to treat hypertension.

[0318] F. Therapeutic Uses of Methylprednisolone

[0319] Methylprednisolone is used to treat a wide range of disorderssuch as asthma and arthritis. In gastroenterology, it is effective inthe treatment of several inflammatory conditions such as ulcerative andmicroscopic colitis, Crohn's disease and autoimmune hepatitis. A newerusage is for reduction of post-traumatic spinal cord edema.

[0320] G. Therapeutic Uses of GP-41 Peptides

[0321] GP-41, an HIV transmembrane protein, can be used to createtherapeutic and diagnostic agents against HIV. For example, antibodiescan be constructed to recognize epitopes of gp41. The structure of thisantibody will provide important information regarding antibody/antigeninteraction, guide chemists in the selection of superior antigenicpeptides for HIV detection, and will provide important information forfuture recombinant experiments with genetically engineered antibodies.

[0322] H. Therapeutic Uses of Blood Brain Barrier (BBB) Peptides

[0323] As BBB peptides can traverse the blood brain barrier throughprotein transduction, these peptides can be covalently linked tocompounds, peptides, antisense peptide nucleic acids or 40-nm ironbeads, or as in-frame fusions with full-length proteins, to allows thesecompounds to enter any cell type in a receptor- andtransporter-independent fashion. This effectively delivers thesecompounds past the blood brain barrier.

[0324] I. Therapeutic Uses of Modified Cell Adhesion (RGD) Peptides

[0325] The RGD peptides of the invention and their derivatives andanalogs find multiple uses including use as a treatment for neoplasticdiseases and inflammatory diseases such as rheumatoid arthritis, lupus.

[0326] 1. Anti-Neoplastic Treatments

[0327] The modified cell adhesion peptides of the invention or theirderivatives or analogs generally will target directly to cancer cellsvia peptide-specific receptors. It has been shown that receptors forthese peptides are expressed at elevated levels on the surface of tumorcells. Thus, the modified peptides or their derivatives or analogs canbe used to preferentially target drugs to metastatic tumor cells.Therefore, the modified cell adhesion peptides or their derivatives oranalogs are useful as agents for the treatment of different types ofcancers such as breast carcinoma, melanoma, and fibrosarcoma.

[0328] The use of an effective amount of modified cell adhesion peptidesor their derivatives or analogs as a treatment for cancer has theadvantage of being more potent than non modified cell adhesion peptides.Since the modified cell adhesion peptides or their derivatives oranalogs are more stable in vivo, smaller amounts of the molecule can beadministered for effective treatment.

[0329] The derivatives and conjugates of the modified cell adhesionpeptides and their analogs may be used in several different ways and toachieve several different ends. As mentioned above, these materials maybe used in place of typical cell adhesion peptide drugs as ananti-adhesive agent. As compared with cell adhesion peptide drugscurrently available, the materials of this invention can reduce clotformation with less side effects and are available for reducing clotformation for a substantially longer time than conventionallyadministered cell adhesion peptide drugs. In addition, the derivatizedcell adhesion peptides of this invention may be utilized (in accordancewith U.S. Pat. Nos. 5,443.827; 5,439,88 and 5,433,940 and PCTapplication number WO/97/01093 which are hereby incorporated byreference) in conjunction with various other anti-adhesive oranti-cancer therapies. Such anti-cancer therapies include the use ofradiation or treatment with antineoplastic agents such as, for example,vinca alkaloids, alkylating agents, doxorubicin, etoposide,methotrexate, tamoxifen, vinblastine, asparaginase, biclutamide,bleomycin, carboplatin, carmustine, chlorambucil, cisplatin, cladribine,cyclophosphamide, cytarabine, dacarbazine, dactinomycin, daunorubisin,docetaxel, floxuridine, fludarabine, fluorouracil, gemcitabine,hydroxyurea, idarubicin, ifosfamide, interferon alpha, irnotecan,leuprolide, mechlorethamine, megestrol, melphalan, mercaptopurine,mitomycin, mitoxantrone, paclitaxel, plicamycin, porfirmer,procarbazine, streptozocin, teniposide, thioguanine, thiotepa,topotecan, trastuzumab, vincristine, vinorelbine, and the like.

[0330] The present invention also provides for a method for treatingcancer in an individual, wherein said method comprises providing anamount of modified cell adhesion peptide sufficient to treat cancer;where the composition contains a modified cell adhesion peptide.

[0331] 2. Treatment Of Inflammatory Disease

[0332] The modified cell adhesion peptides of the invention and theirderivatives and analogs also find use as anti-inflammatories. In oneaspect of the invention, there is provided a method of treating amammalian subject with an abnormality resulting in increasedinflammation of the joints or tissues using the modified cell adhesionpeptides of the invention or their derivatives or analogs. The methodcomprises administering a modified cell adhesion peptide or itsderivative or analog to the subject in an amount sufficient to producean anti-inflammatory effect on the subject. The modified cell adhesionpeptide may be administered intracerebroventriculary, orally,subcutaneously, intramuscularly, or intravenously.

[0333] The peptides of the present invention, their derivatives,analogs, and conjugates can be used to treat acute or chronicinflammatory disorders involving ischemia, infection, tissue swelling,and/or bone and cartilage degradation. Inflammatory disease refers to acondition in which activation of leukocytes leads to an impairment ofnormal physiologic function. Examples of such conditions include acuteand chronic inflammation such as osteoarthritis, sepsis, ARDS, immuneand autoimmune disorders, rheumatoid arthritis, IBD (inflammatory boweldisease), lupus, MS, graft rejection, cirrhosis, sarcoidosis,granulomatous lesions, periodontitis/gingivitis, graft-vs.-host disease,contact dermatitis, and the like. Included among autoimmune disorderswhich may be treated using the present method are chronic activehepatitis, Graves' disease, insulin-dependent diabetes mellitus (typeI), and Hasshimoto's thyroiditis. Included among inflammatory disorderswhich may be treated using the present method are inflammatory braindisease, inflammatory demyelinating disease, inflammatory vasculitis,inflammatory myopathies, osteomyelitis, Crohn's disease and interstitialcystitis. Additional examples of inflammatory diseases includemyocardial diseases, infectious diseases, pulmonary diseases and graftrejection.

[0334] J. Therapeutic Uses of Modified Insulinotropic Peptides Such asGLP-1

[0335] The modified insulinotropic peptides (ITPs) such as GLP-1 of theinvention find multiple uses including use as a treatment for diabetes,a sedative, a treatment of nervous system disorders, use to induce ananxiolytic effect on the CNS, use to activate the CNS, use for postsurgery treatment and as a treatment for insulin resistance.

[0336] 1. Diabetes Treatments

[0337] The modified ITPs of the invention generally will normalizehyperglycemia through glucose-dependent, insulin-dependent andinsulin-independent mechanisms. As such, the modified ITPs are useful asprimary agents for the treatment of type II diabetes mellitus and asadjunctive agents for the treatment of type I diabetes mellitus.

[0338] The use of an effective amount of modified ITPs as a treatmentfor diabetes mellitus has the advantage of being more potent than nonmodified ITPs. Since the modified ITPs are move stable in vivo, smalleramounts of the molecule can be administered for effective tratment. Thepresent invention is especially suited for the treatment of patientswith diabetes, both type I and type II, in that the action of thepeptide is dependent on the glucose concentration of the blood, and thusthe risk of hypoglycemic side effects are greatly reduced over the risksin using current methods of treatment.

[0339] The present invention also provides for a method for treatingdiabetes mellitus in an individual, wherein said method comprisesproviding an amount of modified ITP sufficient to treat diabetes; wherethe composition contains a modified ITP.

[0340] 2. Treatment of Nervous System Disorders

[0341] The modified ITPs of the invention also find use as a sedative.In one aspect of the invention, there is provided a method of sedating amammalian subject with an abnormality resulting in increased activationof the central or peripheral nervous system using the modified ITPs ofthe invention. The method comprises administering a modified ITP to thesubject in an amount sufficient to produce a sedative or anxiolyticeffect on the subject. The modified ITP may be administeredintracerebroventriculary, orally, subcutaneously, intramuscularly, orintravenously. Such methods are useful to treat or ameliorate nervoussystem conditions such as anxiety, movement disorder, aggression,psychosis, seizures, panic attacks, hysteria and sleep disorders.

[0342] In a related aspect, the invention encompasses a method ofincreasing the activity of a mammalian subject, comprising administeringa modified ITP to the subject in an amount sufficient to produce anactivating effect on the subject. Preferably, the subject has acondition resulting in decreased activation of the central or peripheralnervous system. The modified ITPs find particular use in the treatmentor amelioration of depression, schizoaffective disorders, sleep apnea,attention deficit syndromes with poor concentration, memory loss,forgetfulness, and narcolepsy, to name just a few conditions in whicharousal of the central nervous system may be advantageous.

[0343] The modified ITPs of the invention may be used to induce arousalfor the treatment or amelioration of depression, schizoaffectivedisorders, sleep apnea, attention deficit syndromes with poorconcentration, memory loss, forgetfulness, and narcolepsy. Thetherapeutic efficacy of the modified ITP treatment may be monitored bypatient interview to assess their condition, bypsychological/neurological testing, or by amelioration of the symptomsassociated with these conditions. For example, treatment of narcolepsymay be assessed by monitoring the occurrence of narcoleptic attacks. Asanother example, effects of modified ITPs on the ability of a subject toconcentrate, or on memory capacity, may be tested using any of a numberof diagnostic tests well known to those of skill in art.

[0344] 3. Post Surgery Treatment

[0345] The modified ITPs of the invention may be utilized for postsurgery treatments. A patient is in need of the modified ITPs of thepresent invention for about 1-16 hours before surgery is performed onthe patient, during surgery on the patient, and after the patient'ssurgery for a period of not more than about 5 days.

[0346] The modified ITPs of the present invention are administered fromabout sixteen hours to about one hour before surgery begins. The lengthof time before surgery when the compounds used in the present inventionshould be administered in order to reduce catabolic effects and insulinresistance is dependent on a number of factors. These factors aregenerally known to the physician of ordinary skill, and include, mostimportantly, whether the patient is fasted or supplied with a glucoseinfusion or beverage, or some other form of sustenance during thepreparatory period before surgery. Other important factors include thepatient's sex, weight and age, the severity of any inability to regulateblood glucose, the underlying causes of any inability to regulate bloodglucose, the expected severity of the trauma caused by the surgery, theroute of administration and bioavailability, the persistence in thebody, the formulation, and the potency of the compound administered. Apreferred time interval within which to begin administration of themodified ITPs used in the present invention is from about one hour toabout ten hours before surgery begins. The most preferred interval tobegin administration is between two hours and eight hours before surgerybegins.

[0347] Insulin resistance following a particular type of surgery,elective abdominal surgery, is most profound on the first post-operativeday, lasts at least five days, and may take up to three weeks tonormalize Thus, the post-operative patient may be in need ofadministration of the modified ITPs used in the present invention for aperiod of time following the trauma of surgery that will depend onfactors that the physician of ordinary skill will comprehend anddetermine. Among these factors are whether the patient is fasted orsupplied with a glucose infusion or beverage, or some other form ofsustenance following surgery, and also, without limitation, thepatient's sex, weight and age, the severity of any inability to regulateblood glucose, the underlying causes of any inability to regulate bloodglucose, the actual severity of the trauma caused by the surgery, theroute of administration and bioavailability, the persistence in thebody, the formulation, and the potency of the compound administered. Thepreferred duration of administration of the compounds used in thepresent invention is not more than five days following surgery.

[0348] 4. Insulin Resistance Treatment

[0349] The modified ITPs of the invention may be utilized to treatinsulin resistance independently from their use in post surgerytreatment. Insulin resistance may be due to a decrease in binding ofinsulin to cell-surface receptors, or to alterations in intracellularmetabolism. The first type, characterized as a decrease in insulinsensitivity, can typically be overcome by increased insulinconcentration. The second type, characterized as a decrease in insulinresponsiveness, cannot be overcome by large quantities of insulin.Insulin resistance following trauma can be overcome by doses of insulinthat are proportional to the degree of insulin resistance, and thus isapparently caused by a decrease in insulin sensitivity.

[0350] The dose of modified ITPs effective to normalize a patient'sblood glucose level will depend on a number of factors, among which areincluded, without limitation, the patient's sex, weight and age, theseverity of inability to regulate blood glucose, the underlying causesof inability to regulate blood glucose, whether glucose, or anothercarbohydrate source, is simultaneously administered, the route ofadministration and bioavailability, the persistence in the body, theformulation, and the potency.

[0351] K. Therapeutic Uses of Modified Kringle 5 Peptides

[0352] As described earlier, angiogenesis includes a variety ofprocesses involving neovascularization of a tissue including“sprouting”, vasculogenesis, or vessel enlargement. With the exceptionof traumatic wound healing, corpus leuteum formation and embryogenesis,it is believed that the majority of angiogenesis processes areassociated with disease processes and therefore the use of the presenttherapeutic methods are selective for the disease and do not havedeleterious side effects.

[0353] There are a variety of diseases in which angiogenesis is believedto be important, which may be treatable with the modified peptides ofthe invention. These diseases include, but not limited to, inflammatorydisorders such as immune and non-immune inflammation, chronic articularrheumatism and psoriasis, disorders associated with inappropriate orinopportune invasion of vessels such as diabetic retinopathy,neovascular glaucoma, restenosis, capillary proliferation inatherosclerotic plaques and osteoporosis, and cancer associateddisorders, such as solid tumors, solid tumor metastases, angiofibromas,retrolental fibroplasia, hemangiomas, Kaposi sarcoma and the likecancers which require neovascularization to support tumor growth.

[0354] The modified kringle 5 peptides of the invention find use inmethods which inhibit angiogenesis in a diseased tissue amelioratessymptoms of the disease and, depending upon the disease, can contributeto cure of the disease. The modified peptides of the invention are morestable in vivo and, as such, smaller amounts of the modified peptide canbe administered for effective treatment In one embodiment, the inventioncontemplates inhibition of angiogenesis, per se, in a tissue. The extentof angiogenesis in a tissue, and therefore the extent of inhibitionachieved by the present methods, can be evaluated by a variety ofmethod, for detecting ∀₅#₃-immunopositive immature and nascent vesselstructures by immunohistochemistry.

[0355] As described herein, any of a variety of tissues, or organscomprised of organized tissues, can support angiogenesis in diseaseconditions including skin, muscle, gut, connective tissue, joints, bonesand the like tissue in which blood vessels can invade upon angiogenicstimuli.

[0356] In one related embodiment, a tissue to be treated with themodified kringle 5 peptides of the invention is an inflamed tissue andthe angiogenesis to be inhibited is inflamed tissue angiogenesis wherethere is neovascularization of inflamed tissue. In this class the methodcontemplates inhibition of angiogenesis in arthritic tissues, such as ina patient with chronic articular rheumatism, in immune or non-immuneinflamed tissues, in psoriatic tissue and the like.

[0357] The patient treated in the present invention in its manyembodiments is desirably a human patient, although it is to beunderstood that the principles of the invention indicate that theinvention is effective with respect to all mammals, which are intendedto be included in the term “patient.” In this context, a mammal isunderstood to include any mammalian species in which treatment ofdiseases associated with angiogenesis is desirable, particularlyagricultural and domestic mammalian species.

[0358] In another related embodiment, a tissue to be treated with themodified kringle 5 peptides of the invention is a retinal tissue of apatient with diabetic retinopathy, macular degeneration or neovascularglaucoma and the angiogenesis to be inhibited is retinal tissueangiogenesis where there is neovascularization of retinal tissue.

[0359] In an additional related embodiment, a tissue to be treated withthe modified kringle 5 peptides of the invention is a tumor tissue of apatient with a solid tumor, a metastases, a skin cancer, a breastcancer, a hemangioma or angiofibroma and the like cancer, and theangiogenesis to be inhibited is tumor tissue angiogenesis where there isneovascularization of a tumor tissue. Typical solid tumor tissuestreatable by the present methods include lung, pancreas, breast, colon,laryngeal, ovarian, and the like tissues.

[0360] Inhibition of tumor tissue angiogenesis is a particularlypreferred embodiment because of the important role neovascularizationplays in tumor growth. In the absence of neovascularization of tumortissue, the tumor tissue does not obtain the required nutrients, slowsin growth, ceases additional growth, regresses and ultimately becomesnecrotic resulting in killing of the tumor.

[0361] The present invention thus provides for a method of inhibitingtumor neovascularization by inhibiting tumor angiogenesis according tothe present methods using the modified kringle 5 peptides of theinvention. Similarly, the invention provides a method of inhibitingtumor growth by practicing the angiogenesis-inhibiting methods. Themethods are also particularly effective against the formation ofmetastases because (1) their formation requires vascularization of aprimary tumor so that the metastatic cancer cells can exit the primarytumor and (2) their establishment in a secondary site requiresneovascularization to support growth of the metastases.

[0362] In a related embodiment, the invention contemplates the practiceof the method in conjunction with other therapies such as conventionalchemotherapy directed against solid tumors and for control ofestablishment of metastases. The administration of the modified kringle5 peptides of the invention is typically conducted during or afterchemotherapy, although it is preferably to inhibit angiogenesis after aregimen of chemotherapy at times where the tumor tissue will beresponding to the toxic assault by inducing angiogenesis to recover bythe provision of a blood supply and nutrients to the tumor tissue. Inaddition, it is preferred to administer the modified kringle 5 peptidesafter surgery where solid tumors have been removed as a prophylaxisagainst metastases. Insofar as the present methods apply to inhibitionof tumor neovascularization, the methods can also apply to inhibition oftumor tissue growth, to inhibition of tumor metastases formation, and toregression of established tumors using the modified kringle 5 peptidesof the invention.

[0363] Restenosis is a process of smooth muscle cell (SMC) migration andproliferation at the site of percutaneous transluminal coronaryangioplasty which hampers the success of angioplasty. The migration andproliferation of SMC's during restenosis can be considered a process ofangiogenesis which is inhibited by the modified kringle 5 peptides ofthe present invention. Therefore, the invention also contemplatesinhibition of restenosis by inhibiting angiogenesis in a patientfollowing angioplasty procedures. For inhibition of restenosis, themodified kringle 5 peptide is typically administered after theangioplasty procedure for from about 2 to about 28 days, and moretypically for about the first 14 days following the procedure.

[0364] The present method for inhibiting angiogenesis in a tissuecomprises contacting a tissue in which angiogenesis is occurring, or isat risk for occurring, with a composition comprising a therapeuticallyeffective amount of a modified kringle 5 peptide. The dosage ranges forthe administration of the modified kringle 5 peptide depend upon theform of the peptide, and its potency, as described further herein, andare amounts large enough to produce the desired effect in whichangiogenesis and the disease symptoms mediated by angiogenesis areameliorated. The dosage should not be so large as to cause adverse sideeffects, such as hyperviscosity syndromes, pulmonary edema, congestiveheart failure, and the like. Generally, the dosage will vary with theage, condition, sex and extent of the disease in the patient and can bedetermined by one of skill in the art. The dosage can also be adjustedby the individual physician in the event of any complication.

[0365] As angiogenesis inhibitors, such modified kringle 5 peptides areuseful in the treatment of both primary and metastatic solid tumors andcarcinomas of the breast; colon; rectum; lung; oropharynx; hypopharynx;esophagus, stomach; pancreas; liver; gallbladder; bile ducts; smallintestine; urinary tract including kidney, bladder and urothelium;female genital tract including cervix, uterus, ovaries, choriocarcinomaand gestational trophoblastic disease; male genital tract includingprostate, seminal vesicles, testes and germ cell tumors; endocrineglands including thyroid, adrenal, and pituitary; skin includinghemangiomas, melanomas, sarcomas arising from bone or soft tissues andKaposi's sarcoma; tumors of the brain, nerves, eyes, and meningesincluding astrocytomas, gliomas, glioblastomas, retinoblastomas,neuromas, neuroblastomas, Schwannomas and meningiomas; solid tumorsarising from hematopoietic malignancies such as leukemias and includingchloromas, plasmacytomas, plaques and tumors of mycosis fungoides andcutaneous T-cell lymphoma/leukemia; lymphomas including both Hodgkin'sand non-Hodgkin's lymphomas; prophylaxis of autoimmune diseasesincluding rheumatoid, immune and degenerative arthritis; ocular diseasesincluding diabetic retinopathy, retinopathy of prematurity, cornealgraft rejection, retrolental fibroplasia, neovascular glaucoma,rubeosis, retinal neovascularization due to macular degeneration andhypoxia; abnormal neovascularization conditions of the eye; skindiseases including psoriasis; blood vessel diseases including hemagiomasand capillary proliferation within atherosclerotic plaques; Osler-WebberSyndrome; myocardial angiogenesis; plaque neovascularization;telangiectasia; hemophiliac joints; angiofibroma; wound granulation;diseases characterized by excessive or abnormal stimulation ofendothelial cells including intestinal adhesions, Crohn's disease,atherosclerosis, scleroderma and hypertrophic scars (i.e. keloids) anddiseases which have angiogenesis as a pathologic consequence includingcat scratch disease (Rochele minalia quintosa) and ulcers (Helicobacterpylori). Another use is as a birth control agent which inhibitsovulation and establishment of the placenta.

[0366] The modified kringle 5 peptides of the present invention may alsobe useful for the prevention of metastases from the tumors describedabove either when used alone or in combination with radiotherapy and/orother chemotherapeutic treatments conventionally administered topatients for treating angiogenic diseases. For example, when used in thetreatment of solid tumors, the modified kringle 5 peptides of thepresent invention may be administered with chemotherapeutic agents suchas alpha inteferon, COMP (cyclophosphamide, vincristine, methotrexateand prednisone), etoposide, mBACOD (methortrexate, bleomycin,doxorubicin, cyclophosphamide, vincristine and dexamethasone),PRO-MACE/MOPP (prednisone, methotrexate (w/leucovin rescue),doxorubicin, cyclophosphamide, taxol, etoposide/mechlorethamine,vincristine, prednisone and procarbazine), vincristine, vinblastine,angioinhibins, TNP-470, pentosan polysulfate, platelet factor 4,angiostatin, LM-609, SU-101, CM-101, Techgalan, thalidomide, SP-PG andthe like. Other chemotherapeutic agents include alkylating agents suchas nitrogen mustards including mechloethamine, melphan, chlorambucil,cyclophosphamide and ifosfamide; nitrosoureas including carmustine,lomustine, semustine and streptozocin; alkyl sulfonates includingbusulfan; triazines including dacarbazine; ethyenimines includingthiotepa and hexamethylmelamine; folic acid analogs includingmethotrexate; pyrimidine analogues including 5-fluorouracil, cytosinearabinoside; purine analogs including 6-mercaptopurine and6-thioguanine; antitumor antibiotics including actinomycin D; theanthracyclines including doxorubicin, bleomycin, mitomycin C andmethramycin; hormones and hormone antagonists including tamoxifen andcortiosteroids and miscellaneous agents including cisplatin andbrequinar. For example, a tumor may be treated conventionally withsurgery, radiation or chemotherapy and kringle 5 administration withsubsequent kringle 5 administration to extend the dormancy ofmicrometastases and to stabilize and inhibit the growth of any residualprimary tumor.

[0367] L. Therapeutic Uses of Modified Opioid Molecules and AnalgesicAgents

[0368] The derivatives and conjugates of the opioid molecules andanalgesic agents may be used in several different ways and to achieveseveral different ends. These materials may be used in place of typicalantinociceptive agents for alleviating pain. As compared with drugscurrently available, the materials of this invention can alleviate painwithout central mediated side effects or potential of addiction or lossof efficacy, and are available for alleviating pain for a substantiallylonger time than conventionally administered drugs. Opioid derivativesand conjugates of this invention also may be utilized (in accordancewith U.S. Pat. No. 5,482,930) as anti-inflammatory and/oranti-irritation agents or in general to inhibit vascular leakage fromtissues. In addition, as is known in the art, these materials may beused to treat hosts which are or have become tolerant to morphine (or totreat patients undergoing methadone treatment programs), as well astreatment of narcotics withdrawal in general.

[0369] M. Therapeutic Uses of Modified Immuno-Suppressants

[0370] A variety of immuno-suppressant agents such as cyclosporin andderivatives, corticosteroids, sulfasalazine, thalidomide, methotrexate,OKT3, peptide-T, or agents that inhibit T-cell activation or adhesionwould be useful to prior to transplantation to mask immuneresponsiveness and organ rejection. Such agents could be applied at thetime of tissue harvest (e.g. heart, lung, liver harvest) or immediatelyprior to restitution of blood flow in the recipient. Suchimmuno-suppressant agents would prevent the recognition of foreignantigen from the donor tissue that would facilitate short termacceptance and facilitate longer term ability for the host toaccommodate the transplanted organ.

[0371] N. Therapeutic Uses of Modified Antibiotics:

[0372] The modified antibiotics of the invention find use in treatinginfections.

[0373] O. Therapeutic Uses of Modified Antidepressants

[0374] The modified antidepressants of the invention are useful fortreating depression.

[0375] P. Therapeutic Uses of Modified Anti-Viral and Anti-FusogenicPeptides HIV and Anti-HIV Peptides:

[0376] The human immunodeficiency virus (HIV), which is responsible foracquired immune deficiency syndrome (AIDS), is a member of thelentivirus family of retroviruses. There are two prevalent types of HIV,HIV-1 and HIV-2, with various strain of each having been identified. HIVtargets CD-4+ cells, and viral entry depends on binding of the HIVprotein gp41 to CD-4+ cell surface receptors.

[0377] Modified anti-viral or anti-fusogenic peptides of the inventionmay be used as a therapeutic agent in the treatment of patients who aresuffering from HIV infection, and can be administered to patientsaccording to the methods described below and other methods known in theart. Effective therapeutic dosages of the modified peptides may bedetermined through procedures well known by those in the art and willtake into consideration any concerns over potential toxicity of thepeptide.

[0378] The modified peptides can also be administered prophylacticallyto previously uninfected individuals. This can be advantageous in caseswhere an individual has been subjected to a high risk of exposure to avirus, as can occur when individual has been in contact with an infectedindividual where there is a high risk of viral transmission. This can beexpecially advantageous where there is no known cure for the virus, suchas the HIV virus. As a example, prophylactic administration of amodified anti-HIV peptide would be advantageous in a situation where ahealth care worker has been exposed to blood from an HIV-infectedindividual, or in other situations where an individual engaged inhigh-risk activities that potentially expose that individual to the HIVvirus.

[0379] 1. SIV and anti-SIV peptides: Simian immunodeficiency viruses(SIV) are lentiviruses that cause acquired immunodeficiency syndrome(AIDS)-like illnesses in susceptible monkeys. Modified anti-viralpeptides according to the invention can be used for the treatment ofinfected animals or as a prophylactic in a similar fashion as for HIV.

[0380] 2. RSV: Respiratory syncytial virus (RSV) is a respiratorypathogen, especially dangerous in infants and small children where itcan cause bronchiolitis (inflammation of the small air passages) andpneumonia. RSVs are negative sense, single stranded RNA viruses and aremembers of the Paramyxoviridae family of viruses. The route of infectionof RSV is typically through the mucous membranes by the respiratorytract, i.e., nose, throat, windpipe and bronchi and bronchioles.Antiviral peptides according to the invention can be used for preventionand treatment of RSV related diseases.

[0381] 3. HPV: Human parainfluenza virus (HPIV or HPV), like RSV, isanother leading cause of respiratory tract disease, and like RSVs, arenegative sense, single stranded RNA viruses that are members of theParamyxoviridae family of viruses. There are four recognized serotypesof HPIV—HPIV-1, HPIV-2, HPIV-3 and HPIV-4. HPIV-1 is the leading causeof croup in children, and both HPIV-1 and HPIV-2 cause upper and lowerrespiratory tract illnesses. HPIV-3 is more often associated withbronchiolitis and pneumonia. Antiviral peptides according to theinvention can be used for treatment of HPV related diseases.

[0382] 4. MeV: Measles virus (MV or MeV) is an enveloped negative,single-stranded RNA virus belonging to the Paramyxoviridae family ofviruses. Like RSV and HPV, MeV causes respiratory disease, and alsoproduces an immuno-suppression responsible for additional, opportunisticinfections. In some cases, MeV can establish infection of the brainleading to severe neurlogical complications. Antiviral peptidesaccording to the invention can be used for treatment of RSV relateddiseases.

[0383] Q. Therapeutic Uses of Modified Antihistamine Agents

[0384] Modified anthistamine agents find use in treating excesshistamine formed in body tissues including allergic reactions.

[0385] R. Therapeutic Uses of Modified Anti-Angina Agents

[0386] Modified anthi-angina agents find use in treating anginaincluding treatment of choking and suffocating sensations.

[0387] Angina results from insufficient blood supply to the heart, andis often caused by blockages in the arteries that feed the heart musclewith blood (coronary artery stenoses due to atherosclerosis). “Unstable”angina conditions, can develop into acute coronary syndromes (ACS),including myocardial infarction. Antianginal therapies include treatmentwith nitroglycerin and the use of aspirin and heparin.

[0388] Platelet activation and aggregation play an important andessential role in the formation of intracoronary thrombus in acutecoronary syndromes (ACS). Glycoprotein IIb/IIIa receptor inhibitors arecurrently used in connection with heparin and aspirin in ACS.Glycoprotein IIb/IIIa receptor inhibitors block the final step forplatelet aggregation and fibrinogen binding, thus preventing thrombusformation. Tirofiban is a potent, synthetic, non-peptide and specificglycoprotein IIb/IIIa receptor inhibitor and has shown to be welltolerated and to reduce the risk of ischaemic complications in patientswith unstable angina, non-Q-wave myocardial infarction and high-riskpatients undergoing revascularisation when used in combination withaspirin and heparin. Other GP IIb/IIIa receptor inhibitors includeabciximab and eptifibatide.

[0389] S. Use of Modified Thyroxine Molecules

[0390] Thyroxine, an amino acid of the thyroid gland (Merck Index, 1989,9348:1483) and thyroxine analogues are well-known in the art. It is wellestablished in the literature that thyroid hormones, specificallythyroxines T3 and T4, have two distinct types of biological actions: oneon cell metabolism, the second on cell differentiation and development(Jorgensen, 1978, “Thyroid Hormones and Analogues II. Structure-ActivityRelationships,” In: Hormonal Proteins and Peptides, Vol. VI, pp.107-204,C. H. Li, ed., Academic Press, New York). For example, thyroxinesuppresses uptake of iodine by the thyroid (Money et al., 1959, “TheEffect of Various Thyroxine Analogues on Suppression of Iocline-131Uptake by the Rat Thyroid,” Endocrinology 64:123-125) and induces celldifferentiation as studied by tadpole metamorphosis (Money et al., 1958,“The Effect of Change in Chemical Structure of Some Thyroxine Analogueson the Metamorphosis of Rana Pipiens Tadpoles,” Endocrinology 63:20-28).Additionally, thyroxine and certain thyroxine analogues depress growthof non-malignant mouse pituitary thyrotropic tumors (Kumaoka et al.,1960, “The Effect of Thyroxine Analogues on a Transplantable MousePituitary Tumor,” Endocrinology 66:32-38; Grinberg et al., 1962,“Studies with Mouse Pituitary Thyrotropic Tumors. V. Effect of VariousThyroxine Analogs on Growth and Secretion,” Cancer Research 22:835-841).

[0391] The structural requirements of thyroxine and thyroxine analoguesfor metabolic stimulation and induction of cell differentiation are notidentical (see Jorgensen, 1978, “Thyroid Hormones and Analogues II.Structure-Activity Relationships,” In: Hormonal Proteins and Peptides,Vol. VI, p. 150, C. H. Li, ed., Academic Press, New York). For example,Money et al. have found that there is no correlation between suppressionof thyroid iodine uptake and induction of tadpole metamorphosis (Moneyet al., 1958, “The Effect of Change in Chemical Structure of SomeThyroxine Analogues on the Metamorphosis of Rana Pipiens Tadpoles,”Endocrinology 63:20-28). Based on these observations, it was conceivedthat as yet unidentified cellular responses may be altered or induced bycertain thyroxine analogues which do not exhibit either mode of action(metabolic or differentiating) exhibited by thyroxine T3 and T4.

[0392] Deficiency of thyroid activity, whether occurring spontaneouslyor resulting from surgical removal of thyroid gland, thyroiditis, ordecreased function secondary to pituitary degeneration results inclinical hypothyroidism. Whatever the cause, the symptom is treated byreplacement therapy using the modified thyroxine molecules of theinvention.

[0393] The present invention also relates to a method for the treatmentof anemia which is associated with rheumatoid arthritis and of theanemia present in patients having a viral or bacterial infection whereinsymptoms of rheumatoid arthritis are additionally present using themodified thyroxine molecules of the invention.

[0394] According to the invention, the associated anemia which ischaracterized as being moderately hypochromic and normocytic is treatedby administering to a patient in need of treatment a composition forincreasing the thyroxine in the blood stream and thereby increasing theceiling on the number of red cells maturing from the stem cells in theblood stream. The composition can include the presence of ananti-inflammatory agent so as to treat the inflammation present andreduce any pain.

[0395] T. Use of Modified Bronchodialators and Anti-Asthmatic Agents

[0396] Anti-asthmatic agents find use in the treatment of asthma andother lung diseases. Such anti-asthmatic agents include bronchodialatorslike albuterol (Proventil or Ventolin) andmaleimidopropionamyl-1-theobromineacetamide.

[0397] U. Uses of Modified Diagnostic Agents

[0398] The diagnostic agent employed and the vascular protein orproteins targeted will depend upon whether one wishes to diagnosticallyimage the anatomic compartment over an extended period of time, whetherone wishes to preferentially image only a specific cell type orcompartment, or both. Applications for covalently bonding a diagnosticagent of interest to a long-lived vascular protein for diagnosticimaging of the vascular space over an extended period of time arenumerous and include enhancing the ability to detect abnormalities inblood flow throughout the entire mammalian vascular system, includingthe detection of internal injury causing abnormal bleeding or,alternatively, the presence of thromboses. For example, one may wish toimage the vascular space over an extended period of time to detect theeffects of a particular treatment while they occur, i.e., detecting thedisappearance of an embolism, the stoppage of internal bleeding, or thelike.

[0399] Diagnostically imaging the vascular space over an extended periodof time also allows for the detection of various diseases associatedwith the vascular system, i.e., such as arterial blockage in the heart.Thus, diagnostically imaging the vascular system over an extended periodof time may be employed to non-invasively detect a consistently reducedblood flow to the heart. Such a method also provides a means forquantitatively measuring cardiac efficiency and ventricular outputvolume over an extended period of time, i.e., during extended periods ofexercise, or the like.

[0400] Other applications for such a method arise from the ability tonon-invasively visualize anatomical structures of the mammalian vascularsystem and the effects on those anatomical structures over time of theadministration of various drugs, such as vasodilators, vasoconstrictors,or the like. Such may allow for the early detection of developmentalvascular abnormalities, injuries, or the like.

[0401] Additional applications arising from the ability todiagnostically image the vascular space over an extended period of timeinclude functional assessment of the cardiovascular system as routinelyutilized in nuclear medicine for single measurements.

[0402] Applications for preferentially bonding a diagnostic agent ofinterest to a specific protein or proteins present in the vascularsystem so as to diagnostically image only a specific cell type orcompartment are also numerous. For example, having the ability topreferentially direct a diagnostic agent of interest to a specific celltype in the vascular system can allow for the non-invasive and earlydetection of lesions or various tumors associated with the mammalianvascular system by directing the bifunctional anchor molecule to a tumorspecific cell surface protein.

[0403] Additionally, diagnostic agents can be directed to cell surfaceproteins of specific cell types predominantly associated with specificanatomic compartments, allowing one to preferentially diagnosticallyimage such compartments as lymph nodes, Peyer's patches, kidneyglomeruli, liver, pancreas, tonsil, or any other organ to which mobilecells in the vasculature will migrate.

[0404] Other applications for preferentially diagnostic imaging aspecific cell type or compartment of the vascular system includediagnosis and treatment of stenosis or plaque, vascular shuntreendothelialization or shunt failure due to tissue growths, or organrejection due to tissue migration.

[0405] The diagnostic agents of the invetion may be delivered to a localsite via a local delivery device. Delivery devices include catheters,syringes, trocars and endoscopes. Delivery of the agent to a local siteallows imaging of the specific area of delivery. The agents that findparticular use in localized delivery are the non-specific diagnosticagents such as NHS-derivatives.

[0406] The invention can be more clearly illustrated by the followingnon-limiting examples.

EXAMPLE 1 Preparation of Modified RGD Peptide AGYKPEGKRGDAK

[0407] RGD peptide AGYKPEGKRGDAK (SEQ ID NO:1) was synthesized andmodified to include a linking group and a maleimide group according tothe synthesis scheme set forth below.

[0408] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH,Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH,Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Pro-OH,Fmoc-Lys(Boc)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Ala-OH. They weredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Inlast elongation step, the synthesis was then re-automated for theaddition of the 3-maleimidopropionic acid (Step 2). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide was cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 3). The product was purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredmolecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 2 Preparation of Modified RGD Peptid KRGDACEGDSGGPFC

[0409] RGD peptide KRGDACEGDSGGPFC (SEQ ID NO:2) was synthesized andmodified to include a linking group and a maleimide group according tothe synthesis scheme set forth below.

[0410] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Cys(Acm)-OH (C), Fmoc-Phe-OH,Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Cys(Acm)-OH (C),Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Lys(Boc)-OH They were dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). C are cyclizedcysteine. The cyclisation was achieved by cyclization by treatment withTI(TFA)₃ (3 equiv. on 175 ummol scale) when the coupling was paused atlast lysine residue (step 2). After cyclization, In last elongationstep, the synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol. The peptide was cleaved from the resin using 85% TFA/5%TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product was purified by preparative reversedphased HPLC using a Varian (Rainin) preparative binary HPLC system:gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA inCH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μphenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II)at λ 214 and 254 nm to afford the desired protein in >95% purity, asdetermined by RP-HPLC.

EXAMPLE 3 Preparation of Modified RGD Peptide KRGDACEGDSFFPFC

[0411] RGD peptide KRGDACEGDSFFPFC (SEQ ID NO:3) was synthesized andmodified to include a linking group and a maleimide group according tothe synthesis scheme set forth below.

[0412] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Cys(Acm)-OH (C), Fmoc-Phe-OH,Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Ser(tBu)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Cys(Acm)-OH (C),Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Lys(Boc)-OH, Fmoc-AEEA-OH, Fmoc-AEEA-OH, They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). C arecyclized cysteine. The cyclisation was achieved by, cyclization bytreatment with TI(TFA)₃ (3 equiv. on 175 ummol scale) when the couplingwas paused at last lysine residue (step 2). After cyclization, In lastelongation step, the synthesis was then re-automated for the addition ofthe linking group s and the 3-maleimidopropionic acid (Step 3). Betweenevery coupling, the resin was washed 3 times with N,N-dimethylformamide(DMF) and 3 times with isopropanol. The peptide was cleaved from theresin using 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product was purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredpeptidein >95% purity, as determined by RP-HPLC.

EXAMPLE 4 Preparation of Modified GP-41A PeptideYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

[0413] GP-41A peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:4)was synthesized and modified to include a linking group and a maleimidegroup according to the synthesis scheme set forth below.

[0414] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH. They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Inthe last elongation step, the synthesis was automated for the additionof the 3-maleimidopropionic acid (Step 2). Between every coupling, theresin was washed 3 times with N,N-dimethylformamide (DMF) and 3 timeswith isopropanol. The peptide was cleaved from the resin using 85%TFA/5% TIS/5% thioanisole and 5% phenol, followed by precipitation bydry-ice cold Et₂O (Step 3). The product was purified by preparativereversed phased HPLC using a Varian (Rainin) preparative binary HPLCsystem: gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045%TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μphenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II)at λ 214 and 254 nm to afford the desired molecule in >95% purity, asdetermined by RP-HPLC.

EXAMPLE 5 Preparation of Modified GP-41B PeptideYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

[0415] GP-41 B peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ IDNO:5) was synthesized and modified to include a linking group and amaleimide group according to the synthesis scheme set forth below.

[0416] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH.

[0417] They were dissolved in N,N-dimethylformamide (DMF) and, accordingto the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of C₆H₆ CHCl₃ (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for2 h (Step 2). The resin is then washed with CHCl₃ (6×5 mL), 20% AcOH inDCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

[0418] In the last elongation step, the synthesis was automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide was cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product was purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredmolecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 6 Preparation of Modified GP-41C PeptideYTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF

[0419] GP-41C peptide YTSLIHSLIEESQNQQEKNEQELLELDKWASLWNWF (SEQ ID NO:6)was synthesized and modified to include a linking group and a maleimidegroup according to the synthesis scheme set forth below.

[0420] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Phe-OH, Fmoc-Trp(Boc)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Trp(Boc)-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-Ala-OH, Fmoc-Trp(Boc)-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Lys(Boc)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Gln(Trt)-OH,Fmoc-Asn(Trt)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH, Fmoc-Ser(tBu)-OH,Fmoc-His(Boc)-OH, Fmoc-Ile-OH, Fmoc-Leu-OH Fmoc-Ser(tBu)-OH,Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)-OH, They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

[0421] The selective deprotection of the Lys (Aloc) group is performedmanually and accomplished by treating the resin with a solution of 3 eqof Pd(PPh₃)₄ dissolved in 5 mL of C₆H₆ CHCl₃ (1:1): 2.5% NMM (v:v): 5%AcOH (v:v) for 2 h (Step 2). The resin is then washed with CHCl₃ (6×5mL), 20% AcOH in DCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).

[0422] In the last elongation step, the synthesis was automated for theaddition of the Fmoc-AEEA-OH and finally 3-maleimidopropionic acid (Step2). Between every coupling, the resin was washed 3 times withN,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptidewas cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 3). Theproduct was purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm to afford thedesired molecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 7 Preparation of Modified RSV PeptideVYPSDEYDASISQVNEEINQALAYIRKADELLENV

[0423] RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ ID NO:7) wassynthesized and modified to include a linking group and a maleimidegroup according to the synthesis scheme set forth below.

[0424] Solid phase peptide synthesis on a 100 μmole scale is performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Rink Amide MBHA. The following protected amino acids aresequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Val-OH,Fmoc-Asn(Trt)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH,Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH,Fmoc-Leu-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH,Fmoc-Val-OH, Fmoc-Gln(Trt)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH, Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH. They are dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1).

[0425] The amino group of the final amino acid is acetylated usingAcetic Acid activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). The selective deprotection ofthe Lys (Aloc) group is performed manually and accomplished by treatingthe resin with a solution of 3 eq of Pd(PPh₃)₄ dissolved in 5 mL ofCHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washedwith CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF(6×5 mL). The synthesis is then re-automated for the addition of the3-maleimidopropionic acid (Step 3).

[0426] Between every coupling, the resin is washed 3 times withN,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptide iscleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct is purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm to afford thedesired molecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 8 Preparation of Modified RSV PeptideVYPSDEYDASISQVNEEINQALAYIRKADELLENV

[0427] RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ ID NO:8) wassynthesized and modified to include a linking group and a maleimidegroup to produce the modified peptide depicted below.

[0428] Solid phase peptide synthesis on a 100 μmole scale is performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Rink Amide MBHA. The following protected amino acids aresequentially added to resin: Fmoc-Val-OH, Fmoc-Asn(Trt)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Gln(Trt)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH,Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH,Fmoc-Lys(Aloc)-OH. They are dissolved in N,N-dimethylformamide (DMF)and, according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup is achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The amino group ofthe final amino acid is acetylated using Acetic Acid, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA).The selective deprotection ofthe Lys (Aloc) group is performed manually and accomplished by treatingthe resin with a solution of 3 eq of Pd(PPh₃)₄ dissolved in 5 mL ofCHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). The resin is then washedwith CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM (6×5 mL), and DMF(6×5 mL). The synthesis is then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resin iswashed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol. The peptide is cleaved from the resin using 85% TFA/5%TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-icecold Et₂O (Step 4). The product is purified by preparative reversedphased HPLC using a Varian (Rainin) preparative binary HPLC system:gradient elution of 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA inCH₃CN (B) over 180 min at 9.5 mL/min using a Phenomenex Luna 10μphenyl-hexyl, 21 mm×25 cm column and UV detector (Varian Dynamax UVD II)at λ214 and 254 nm to afford the desired molecule in >95% purity, asdetermined by RP-HPLC.

EXAMPLE 9 Preparation of Modified RSV PeptideVYPSDEYDASISQVNEEINQALAYIRKADELLENV

[0429] RSV peptide VYPSDEYDASISQVNEEINQALAYIRKADELLENV (SEQ ID NO:9) wassynthesized and modified to include a linking group and a maleimidegroup according to the synthesis scheme set forth below.

[0430] Solid phase peptide synthesis on a 100 μmole scale is performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Rink Amide MBHA. The following protected amino acids aresequentially added to resin: Fmoc-Val-OH, Fmoc-Asn(Trt)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Leu-OH, Fmoc-Glu(tBu)-OH,Fmoc-Asp(tBu)-OH, Fmoc-Ala-OH, Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Ile-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Ala-OH, Fmoc-Leu-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Ile-OH, Fmoc-Glu(tBu)-OH,Fmoc-Glu(tBu)-OH, Fmoc-Asn(Trt)-OH, Fmoc-Val-OH, Fmoc-Gln(Trt)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ile-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ala-OH,Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Glu(tBu)-OH, Fmoc-Asp(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Pro-OH, Fmoc-Tyr(tBu)-OH, Fmoc-Val-OH. They aredissolved in N,N-dimethylformamide (DMF) and, according to the sequence,activated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Theamino group of the final amino acid is acetylated using Acetic Acidactivated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).Theselective deprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredmolecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 10 Preparation of Modified GLP-1 PeptideHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK

[0431] GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK (SEQ ID NO:10) wassynthesized and modified to include a linking group and a maleimidegroup according to the synthesis scheme set forth below.

[0432] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Gly-OH, Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH,Fmoc-Trp(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH,Fmoc-Glu(tBu)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH,Fmoc-Gln(Trt)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH,Fmoc-Tyr(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH,Fmoc-Asp(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH,Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH,Fmoc-His(Boc)-OH, The following protected amino acids were sequentiallyadded to resin: They were dissolved in N,N-dimethylformamide (DMF) and,according to the sequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of C₆H₆ CHCl₃ (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for2 h (Step 2). The resin is then washed with CHCl₃ (6×5 mL), 20% AcOH inDCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL).The synthesis was thenre-automated for the addition of the 3-maleimidopropionic acid (Step 3).Between every coupling, the resin was washed 3 times withN,N-dimethylformamide (DMF) and 3 times with isopropanol. The peptidewas cleaved from the resin using 85% TFA/5% TIS/5% thioanisole and 5%phenol, followed by precipitation by dry-ice cold Et₂O (Step 4). Theproduct was purified by preparative reversed phased HPLC using a Varian(Rainin) preparative binary HPLC system: gradient elution of 30-55% B(0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column andUV detector (Varian Dynamax UVD II) at λ 214 and 254 nm to afford thedesired molecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 11 Preparation of Modified GLP-1 PeptideHAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK

[0433] GLP-1 peptide HAEGTFTSDVSSYLEGQAAKEFIAWLVKGRK (SEQ ID NO:11) wassynthesized and modified to include a linking group and a maleimidegroup, as described below.

[0434] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin: Fmoc-Arg(Pbf)-OH, Fmoc-Gly-OH,Fmoc-Lys(Boc)-OH, Fmoc-Val-OH, Fmoc-Leu-OH, Fmoc-Trp(Boc)-OH,Fmoc-Ala-OH, Fmoc-Ile-OH, Fmoc-Phe-OH, Fmoc-Glu(tBu)-OH,Fmoc-Lys(Aloc)-OH, Fmoc-Ala-OH, Fmoc-Ala-OH, Fmoc-Gln(Trt)-OH,Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Leu-OH, Fmoc-Tyr(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Val-OH, Fmoc-Asp(tBu)-OH,Fmoc-Ser(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Phe-OH, Fmoc-Thr(tBu)-OH,Fmoc-Gly-OH, Fmoc-Glu(tBu)-OH, Fmoc-Ala-OH, Fmoc-His(Boc)-OH, Thefollowing protected amino acids were sequentially added to resin: Theywere dissolved in N,N-dimethylformamide (DMF) and, according to thesequence, activated usingO-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). Removal of the Fmoc protectinggroup was achieved using a solution of 20% (V/V) piperidine inN,N-dimethylformamide (DMF) for 20 minutes (step 1). The selectivedeprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of C₆H₆ CHCl₃ (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for2 h (Step 2). The resin is then washed with CHCl₃ (6×5 mL), 20% AcOH inDCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). The linking groupFmoc-AEEA-OH was added and then the Fmoc was removed in the usualfashon. This procedure was redone to add a second AEEA linking group.

[0435] The synthesis was then re-automated for the addition of the3-maleimidopropionic acid (Step 3). Between every coupling, the resinwas washed 3 times with N,N-dimethylformamide (DMF) and 3 times withisopropanol. The peptide was cleaved from the resin using 85% TFA/5%TIS/5% thioanisole and 5% phenol, followed by precipitation by dry-icecold Et₂O (Step 4).

[0436] The product was purified by preparative reversed phased HPLCusing a Varian (Rainin) preparative binary HPLC system: gradient elutionof 30-55% B (0.045% TFA in H₂O (A) and 0.045% TFA in CH₃CN (B)) over 180min at 9.5 mL/min using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cmcolumn and UV detector (Varian Dynamax UVD II) at λ 214 and 254 nm toafford the desired molecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 12 Preparation of Modified K5 Peptide PRKLYDYK

[0437] K5 peptide PRKLYDYK (SEQ ID NO:12) was synthesized and modifiedto include a linking group and a maleimide group according to thesynthesis scheme set forth below.

[0438] Using automated peptide synthesis, the following protected aminoacids were sequentially added to Rink Amide MBHA resin (0.48 mmol/mg)(250 μmol scale): Fmoc-Lys(Aloc)OH, Fmoc-Tyr(tBu)OH, Fmoc-Asp(tBu)-OH,Fmoc-Tyr(tBu)OH, Fmoc-Leu-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Pro-OH. Each coupling was accomplished using 2 equivalents of aminoacid, 1 equivalent HBTU, and 2 equivalents DIEA and performed twice for30 min. The Fmoc group of the N-terminal amino acid (Pro) was removedusing 20% piperidine/DMF (3×10min).

[0439] The resin was subsequently washed with 6×4 mL DMF, 3×3 mL EtOHand 6×4 mL DMF. Acetylation of the N-terminus was accomplished manuallyon the Symphony by adding 4 mL of 15 equivalents HOAc, 2 equivalentsDIEA and 4 equivalents HBTU in DMF. Acetic capping was performed 2×30min. The resin was subsequently washed with 3×4 mL CH₂Cl₂, 6×4 mL 0.5%DIEA/CH₂Cl₂, 3×4 mL EtOH and 6×4 mL DMF Selective deprotection of theLys(Aloc) group was performed manually on the Symphony by treating theresin with a solution of 3 equivalents of Pd(PPh₃)₄ dissolved in 5 mL ofCHCl₃:Benzene (1:1) with 2.5% NMM (v/v) and 5% HOAc for 2 h. The resinwas then washed with CHCl₃ (6×5 mL), 0.5% DIEA in CH₂Cl₂ (6×5 mL), 0.02M sodium diethylthiocarbamate in DMF (6×5 mL), EtOH (3×4 mL) and DMF(6×5 mL).

[0440] Coupling of 3-maleimidoproprionic acid (MPA) was performed byresuming automation on the Symphony, which involves delivery of 2equivalents of MPA, 2 equivalents DIEA and 1 equivalent HBTU to thereaction vessel. The coupling was carried out twice at 30 min. Washingwas conducted using 6×4 mL DMF, 3×3 mL EtOH and 6×4 mL DMF. Cleavagefrom the resin was performed by automation using 10 mL of the followingcleavage mixture: 85% TFA/5% triisopropyl silane/5% thioanisol/5%phenol. After the peptide was cleaved from the resin for 2 hrs, theresin was washed with TFA and CH₂Cl₂. The combined cleavage and washingliquors concentrated to 1-2 mL using a rotovap with mild heating (30°C.) and the peptide was precipitated with Et₂O. The precipitate wascollected by filtration using a SPPS manifold and washed with 10 mL ofethyl acetate and 30 mL of Et₂O. The precipitate was subsequentlydissolved in 10 mL of water containing 5% acetonitrile (0.04% TFA) inwater (0.04% TFA) for chromatographic purification.

EXAMPLE 13 Preparation of Modified K5 Peptide RNPDGDVGGPWAWTTAPRKLYDY

[0441] K5 peptide RNPDGDVGGPWAWTTAPRKLYDY (SEQ ID NO:13) was synthesizedand modified to include a linking group and a maleimide group accordingto the synthesis scheme set forth below.

[0442] Using automated peptide synthesis, the following protected aminoacids were sequentially added to Rink Amide MBHA resin (0.48 mmol/mg)(100 μmol scale): Fmoc-Tyr(tBu)OH, Fmoc-Asp(tBu)-OH, Fmoc-Tyr(tBu)OH,Fmoc-Leu-OH Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Pro-OH,Fmoc-Asn(Trt)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tyr(tBu)OH,Fmoc-Ala-OH, Fmoc-Trp-OH, Fmoc-Pro-OH, Fmoc-Gly-OH, Fmoc-Gly-OH,Fmoc-Val-OH, Fmoc-Asp(tBu)-OH, Fmoc-Gly-OH, Fmoc-Asp(tBu)-OH,Fmoc-Pro-OH, Fmoc-Asn(Trt)-OH, Fmoc-Arg(Pbf)-OH, MPA.

[0443] Each coupling was accomplished using 5 equivalents of amino acid,1 equivalent HBTU, and 2 equivalents DIEA and performed twice for 30min. Cleavage from the resin was performed by automation using 10 mL ofthe following cleavage mixture: 85% TFA/5% triisopropyl silane/5%thioanisol/5% phenol. After the peptide was cleaved from the resin for 2hrs, the resin was washed with TFA and CH₂Cl₂.

[0444] The combined cleavage and washing liquors concentrated to 1-2 mLusing a rotovap with mild heating (30° C.) and the peptide wasprecipitated with Et₂O. The precipitate was collected by filtrationusing a SPPS manifold and washed with 10 mL of ethyl acetate and 30 mLof Et₂O. The precipitate was subsequently dissolved in 10 mL of watercontaining 5% acetonitrile (0.04% TFA) in water (0.04% TFA) forchromatographic purification. Purification of all the peptides wasperformed using a Phenomenex Luna 10μ phenyl-hexyl, 21 mm×250 mm columnequilibrated with a water/TFA mixture (0.045% TFA in H₂O; Solvent A).

[0445] Elution was achieved at 18 mL/min by running a 10-30%acetonitrile gradient over 60 min (0.045% TFA in CH₃CN; Solvent B).Peptides were detected by UV absorbance (Varian Dynamax UVD II) at 214and 254 nm. Fractions were collected in 9 mL aliquots. Fractionscontaining the desired product were identified by mass after directinjection onto LC/MS. The selected fractions were subsequently analyzedby analytical HPLC (10-40% solvent B over 20 min; Phenomenex Luna 5μphenyl-hexyl, 10 mm×250 mm column, 0.5 mL/min) to identify fractionswith ≧95% purity for pooling. The pool was freeze-dried using dry iceand acetone and subsequently lyophilized for at least 2 days to yield awhite powder.

EXAMPLE 14 Preparation of Modified BBB Peptide YGRKKRRQRRRL

[0446] BBB peptide YGRKKRRQRRRL (SEQ ID NO:14) was synthesized andmodified to include a linking group and a maleimide group according tothe synthesis scheme set forth below.

[0447] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Afterthe tyrosine deprotection, Biotin was anchored at the N-terminus viaregular activation and coupling conditions. The selective deprotectionof the Lys (Aloc) group is performed manually and accomplished bytreating the resin with a solution of 3 eq of Pd(PPh₃)₄ dissolved in 5mL of C₆H₆ CHCl₃ (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for 2 h (Step 2).The resin is then washed with CHCl₃ (6×5 mL), 20% AcOH in DCM (6×5 mL),DCM (6×5 mL), and DMF (6×5 mL).The synthesis was then re-automated forthe addition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide was cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product was purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredmolecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 15 Preparation of Modified BBB Peptide YGRKKRRQRRRL

[0448] BBB peptide YGRKKRRQRRRL (SEQ ID NO:15) was synthesized andmodified to include a linking group and a maleimide group according tothe synthesis scheme set forth below.

[0449] Solid phase peptide synthesis on a 100 μmole scale was performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids weresequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Arg(Pbf)-OH,Fmoc-Gly-OH, Fmoc-Tyr(tBu)-OH,They were dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group was achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Theselective deprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of C₆H₆ CHCl₃ (1:1): 2.5% NMM (v:v): 5% AcOH (v:v) for2 h (Step 2). The resin is then washed with CHCl₃ (6×5 mL), 20% AcOH inDCM (6×5 mL), DCM (6×5 mL), and DMF (6×5 mL). After the alocdeprotection, Biotin was anchored at the ε-N terminal of the deprotectedlysine via regular activation and coupling conditions. The Fmoc removalof the N-terminus was then achieved with standard conditions. Thesynthesis was then re-automated for the addition of the3-maleimidopropionic acid at the end terminus (Step 3). Between everycoupling, the resin was washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide was cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product was purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B)) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredmolecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 16 Preparation of Modified dynorphin Peptide YGGFLRRIRPKLK

[0450] Dynorphin peptide YGGFLRRIRPKLK (SEQ ID NO:16) was synthesizedand modified to include a linking group and a maleimide group accordingto the synthesis scheme set forth below.

[0451] Solid phase peptide synthesis on a 100 μmole scale is performedusing manual solid-phase synthesis, a Symphony Peptide Synthesizer andFmoc protected Ramage Resin. The following protected amino acids aresequentially added to resin: Fmoc-Lys(Aloc)-OH, Fmoc-Leu-OH,Fmoc-Lys(Boc)-OH, Fmoc-Pro-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Ile-OH,Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Leu-OH, Fmoc-Phe-OH,Fmoc-Gly-OH, Fmoc-Gly-OH, Boc-Tyr(tBu)-OH. They are dissolved inN,N-dimethylformamide (DMF) and, according to the sequence, activatedusing O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). Removal ofthe Fmoc protecting group is achieved using a solution of 20% (V/V)piperidine in N,N-dimethylformamide (DMF) for 20 minutes (step 1). Theamino group of the final amino acid is acetylated using Acetic Acidactivated using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyl-uroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA).Theselective deprotection of the Lys (Aloc) group is performed manually andaccomplished by treating the resin with a solution of 3 eq of Pd(PPh₃)₄dissolved in 5 mL of CHCl₃:NMM:HOAc (18:1:0.5) for 2 h (Step 2). Theresin is then washed with CHCl₃ (6×5 mL), 20% HOAc in DCM (6×5 mL), DCM(6×5 mL), and DMF (6×5 mL). The synthesis is then re-automated for theaddition of the 3-maleimidopropionic acid (Step 3). Between everycoupling, the resin is washed 3 times with N,N-dimethylformamide (DMF)and 3 times with isopropanol. The peptide is cleaved from the resinusing 85% TFA/5% TIS/5% thioanisole and 5% phenol, followed byprecipitation by dry-ice cold Et₂O (Step 4). The product is purified bypreparative reversed phased HPLC using a Varian (Rainin) preparativebinary HPLC system: gradient elution of 30-55% B (0.045% TFA in H₂O (A)and 0.045% TFA in CH₃CN (B) over 180 min at 9.5 mL/min using aPhenomenex Luna 10μ phenyl-hexyl, 21 mm×25 cm column and UV detector(Varian Dynamax UVD II) at λ 214 and 254 nm to afford the desiredmolecule in >95% purity, as determined by RP-HPLC.

EXAMPLE 172-[2-[4-[4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide.(Modified Cetirizine)

[0452] A mixture of 1-[(4-chlorophenylmethyl]-piperazine 1, methyl(2-chloroethoxy)-acetate 2 and sodium carbonate in anydrous xylene isheated under reflux with good stirring as indicated in the schematicbelow. The reaction mixture is then cooled and filtered and the solid iswashed with benzene, the washed solid being discarded. The filtrate isevaporated to dryness and the evaporation residue is purified bychromatography on a column of silica (eluent: chloroform:methanol 97:3v/v). This generated methyl2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxy]-acetate 3.The compound is dissolved in of absolute ethanol. 1 N ethanolic solutionof potassium hydroxide is then added thereto and the reaction mixture isheated under reflux for 4 hours. It is cooled and the precipitateremoved by filitration, after washing with diethyl ether. The filtrateis evaporated to dryness and the evaporation residue is triturated withdiethyl ether and left to crystallize. The compound potassium2-[2-[4-[(4-chlorophenyl)phenylmethyl]-1-piperazinyl]ethoxy]-acetate isthen obtained. The potassium salt is dissolved in water and adjustedwith 10% hydrochloric acid to a pH of 4. The solution is extracted withchloroform and the organic phase is dried over anhydrous magnesiumsulfate, whereafter it is evaporated to dryness. The evaporation residueis triturated with diethyl ether and left to crystallize to produce2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-acetic acid4. 2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-aceticacid 4 is then placed in DMF and activated with andO-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). To the reaction mixture isadded 3-maleimidopropylamine. The reaction is stirred for 3 hours. Theorganic phase is then washed with water and brine, dried over MgSO₄,triturated with cold ether and left to crystallize. This last stepgenerated 52-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide,a modified antihistamine molecule.

EXAMPLE 1811-(N-maleimidopropionyl4-piperidylidene)-8-chloro-6,11-dihydro-5H-benzo-[5.6]-cyclohepta-[1,2-b]-pyridine(Modified Loratidine).

[0453]11-(N-8-chloro-4-piperidylidene)-6,11-dihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-pyridine1 is placed is placed in DMF and activated with andO-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA) as indicated in the schematicbelow. To the reaction mixture is added 3-maleimidopropionic acid. Thereaction is stirred for 3 hours. The organic phase is then washed withwater and brine, dried over MgSO₄, chromatographied triturated with coldether and left to crystallize to generate2,11-(N-maleimidopropionyl-4-piperidylidene)-8-chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-pyridine,a modified antihistamine molecule.

EXAMPLE 19 Modified Tirofiban

[0454] A four-neck round bottom flask equipped with a mechanicalstirrer, condenser, nitrogen inlet, HCl trap, heating unit and athermometer probe is purged with nitrogen overnight and then chargedwith L-tyrosine 1, CH₃CN,N,O-bis-trimethylsilyl-trifluoromethyl-acetamide. The suspension isheated to gentle reflux for 2 h. The resulting clear solutionO,O′-bis-trimethylsilyl-(L)-tyrosine 2, is cooled pyridine and n-BuSO₂Clare slowly added over 30 minutes as indicated in the schematic below.The reaction mixture is then stirred at room temperature. Almost all thesolvent is removed in a batch concentrator, and the resulting oilyresidue is treated with 15%/KHSO4 and stirred vigorously for 1 hour. Themixture is extracted with i-propyl acetate. The combined organic layeris treated with Ecosorb TM S-402 and stirred at room temperatureovernight. Ecosorb TM is removed by filtration and the filter cake iswashed with i-propyl acetate. The filtrate is evaporated to dryness andthe resulting yellow oil is dissolved in hot EtOAc. Hexane is addedslowly to the stirring solution and the resulting slurry is stirred atroom temperature overnight. The solid is collected by filtration and thefilter cake is washed with EtOAc/hexane. After drying under vacuum isobtained as a white solid.

[0455] To a four-neck round bottom flask equipped with a mechanicalstirrer, condenser, nitrogen inlet and a thermometer probe is chargedN-n-butanesulfonyl-(L)-tyrosine 3,4-(4-pyridinyl)-butyl chloride HCl 4and DMSO. With vigorous stirring, 3 N aq. KOH is added over 15 min.

[0456] The temperature is maintained in the 30-40° C. range for thisoperation using cooling water. Potassium iodide is added, and themixture is heated for 36 h. After cooling to room temperature, themixture is diluted with 0.25 N NaOH and extracted once with t-butylmethyl ether. The aqueous layer is treated with Ecosorb S-402 and NucharSA and the resulting mixture is mechanically stirred for 1 h. Themixture is filtered through a coarse-porosity sintered funnel and thefiltered cake is washed with water. The combined filtrate is placed in avessel equipped with a pH meter probe and a mechanical stirrer. Withvigorous stirring, NaCl is added, stirred for 30 min, and then 50% aq.acetic acid wash added until pH 4.80, and stirring continued for 2-3 h.The resulting slurry is filtered through a coarse-porosity sinteredfunnel, and the cake is washed with water. The crude product is driedunder house-vacuum under a positive nitrogen pressure to give beigesolid 5 having a wt % purity of 95%.

[0457] Selective hydrogenation of the pyridine ring to piperidine ringis accomplished by using 5 wt % of 10% Pd/C in AcOH at 60C to give thetarget product cleanly without reduction of the phenolic ring.Filtration of the reaction mixture, evaporation of acetic acid followedby crystallizing the product 6 from 6% AcOH/water.

[0458] To a RB flask equipped with a thermometer probe and additionfunnel is charged the crude and 0.25 N NaOH. After complete dissolution,the solution is cooled to room temperature, and adjusted to pH 7 by slowadditon of 1 N HCl .The solution is further brought down to pH 5.5 byslow addition of 0.5 N HCl. Stirring is continued for 1 h, then theslurry is filtered through a coarse funnel padded with a sheet ofshark-skin paper and a polypropylene pad (10 mu m) and the cake iswashed with water. The solid is dried under house vacuum with nitrogensweep to give a beige solid. The compound is then placed in DMF andactivated with and O-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To thereaction mixture is added 3-maleimidopropylamine. The reaction isstirred for 3 hours. The organic phase is then washed with water andbrine, dried over MgSO₄, triturated with cold ether and left tocrystallize to generate the modifed tirofiban 7. Tirofiban is ananti-angina agent.

EXAMPLE 20N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropionilamide(modifed-Enalapril)

[0459] Ethyl 2-oxo-4-phenylbutyrate 1 and L-alanyl-L-proline 2 aredissolved in a 1:1 ethanol-water solvent as indicated in the schematicbelow. A solution of sodium cyanoborohydride in ethanol-water is addeddropwise at room temperature over the course of two hours. When reactionis complete, the product is absorbed on strong acid ion-exchange resinand eluted with 2% pyridine in water. The product-rich cuts are freezedried to give crudeN-(1-ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-proline 4 and thecompound is purified by chromatography to yield the desired isomer. Thecompound 4 is then placed in DMF and activated with andO-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluronium hexafluorophosphate(H BTU) and Diisopropylethylamine (DIEA). To the reaction mixture isadded 3-maleimidopropylamine. The reaction is stirred for 3 hours. Theorganic phase is then washed with water and brine, dried over MgSO₄,triturated with cold ether and left to crystallize to produce 5N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropionilamide,a modified anti-hypertensive agent.

EXAMPLE 21Maleimidopropynamyl-ε-(3,4,5-trimethoxybenz-amido)-caproicamide(Modified-Capobenic Acid)

[0460] 3,4,5-trimethoxybenzoyl chloride 1 is added along withamino-hexanoic acid 2 in a solution of 1N NaOH as indicated in theschematic below. The resulting solution is preferably treated with charto decolorize it, the char is filtered, and the filtrate neutralizedwith dilute HCl to Congo red indicator end-point. The resultingprecipitate is separated by filtration washed with water, dried, thenrecrystallized from ethanol to genarate 3. The compound is then placedin DMF and activated with andO-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluronium hexafluorophosphate(H BTU) and Diisopropylethylamine (DIEA). To the reaction mixture isadded 3-maleimidopropylamine. The reaction is stirred for 3 hours. Theorganic phase is then washed with water and brine, dried over MgSO₄,triturated with cold ether and left to crystallize in order to produce 4Maleimidopropynamyl-ε-(3,4,5-trimethoxybenz-amido)-caproicamide, ananti-arrhthymetic agent

EXAMPLE 22 Maleimidopropionamyl-1-theobromineacetamide (Modified1-theobromineacetic acid)

[0461] 1-theobromineacetic acid 1 is placed in DMF and activated withO-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA) as indicated in the schematicbelow. To the reaction mixture is added 3-maleimidopropylamine. Thereaction is stirred for 3 hours. The organic phase is then washed withwater and brine, dried over MgSO₄, chromatographied, triturated withcold ether and left to crystallize to produce 2Maleimidopropionamyl-1-theobromineacetamide, a modifed bronchodilator.

EXAMPLE 23 4-anilino-1-(2-phenethyl)piperidin (Modified-Fentanyl)

[0462] 1-phenylethyl-4-piperidone 1 was placed in 1,2, dichloroethanealong with aniline 2, sodiumcyanoborohydride and it is refluxed for 18hours. The reaction is then cooled to RT and the reaction is extractedwith brine to generate 3 as indicated in the schematic below. FinallyThe compound is then placed in DMF and activated with andO-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). To the reaction mixture isadded 3-maleimidopropionic acid. The reaction is stirred for 3 hours.The organic phase is then washed with water and brine, dried over MgSO₄,triturated with cold ether and left to crystallize to generate 4, amodified pain killer (opioid molecule).

EXAMPLE 24 Maleimidopropamyl2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamide(Modified-Loxoprofen).

[0463] Ethyl 2 cyclopentanonecarboxylate 1 and ethyl2-(4-iodomethylphenyl)propionate 2 are placed in N,N,dimethylformamidealong with potassium hydroxyde as indicated in the schematic below. Thesolution is stirred at room temperature for 5 hours and at 50° C. for 1hour. The reaction is cooled and acidified with acetic acid andN,N,dimethylformamide is removed by vacuum. The residue is extractedwith ether and the organic phase is washed with water and dried onMg₂SO₄ to afford 3 Finally, the compound 3 is then placed in DMF andactivated with and O-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA). To thereaction mixture is added 3-maleimidopropylamine. The reaction isstirred for 3 hours. The organic phase is then washed with water andbrine, dried over MgSO₄, chromatographied, triturated with cold etherand left to crystallize to generate 4 Maleimidopropamyl2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamide to produce themodified anti-inflammatory agent.

EXAMPLE 25 N-maleimidopropionyl-N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine (Modified Fluoxetine)

[0464] β-dimethylaminopropiophenone hydrochloride 1 is converted to thecorresponding free base by the action of aqueous sodium hydroxide. Theliberated free base is taken up in ether, the ether layer separated anddried, and the ether removed therefrom in vacuo. The residual oilcomprising β-dimethylaminopropiophenone is dissolved in tetrahydrofuran,and the resulting solution added in dropwise fashion with stirring to asolution of diborane in tetrahydrofuran. The reaction mixture is stirredovernight at room temperature. Next, aqueous hydrochloric acid is addedto decompose any excess diborane present. The tetrahydrofuran is removedby evaporation. The acidic solution is extracted twice with benzene, andthe benzene extracts are discarded. The acidic solution is then madebasic with an excess of 5 N aqueous sodium hydroxide. The basic solutionis extracted three times with benzene. The benzene extracts areseparated and combined, and the combined extracts washed with asaturated aqueous sodium chloride and then dried to produce 2. Asolution containing N,N,-dimethyl 3-phenyl-3-hydroxypropylamine 2 inchloroform is saturated with dry gaseous hydrogen chloride. Thionylchloride is then added to the chloroform solution at a rate sufficientto maintain reflux. The solution is refluxed an additional 5 hours.Evaporation of the chloroform and other volatile constituents in vacuoyielded N,N-dimethyl 3-phenyl-3-chloropropylamine hydrochloride 3 whichis collected by filtration, and the filter cake washed twice withacetone. P-trifluoromethylphenol 4, solid sodium hyroxide and methanolare placed in a round-bottom flask equipped with magnetic stirrer,condenser and drying tube. The reaction mixture is stirred until thesodium hydroxide had dissolved. Next, N,N-dimethyl3-phenyl-3-chloropropylamine hydrochloride is added. The resultingreaction mixture is refluxed for about 5 days and then cooled. Themethanol was then removed by evaporation, and the resulting residuetaken up in a mixture of ether and 5 N aqueous sodium hydroxide. Theether layer is separated and washed twice with 5 N aqueous sodiumhydroxide and three times with water. The ether layer is dried, and theether removed by evaporation in vacuo to yield as a residue N,N-dimethyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 5. A solutioncontaining cyanogen bromide in benzene and toluene is placed in athree-neck round-bottom flask equipped with thermometer, additionfunnel, drying tube and inlet tube for nitrogen. The solution is cooledand nitrogen gas is bubbled thru the solution. Next, a solution ofN,N-dimethyl 3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 5dissolved in benzene is added in dropwise fashion. The temperature ofthe reaction mixture is allowed to rise slowly to room temperature, atwhich temperature stirring is continued overnight while stillmaintaining a nitrogen atmosphere.The reaction mixture is washed twicewith water, once with 2 N aqueous sulfuric acid and then with wateruntil neutral. The organic layer is dried, and the solvents removedtherefrom by evaporation in vacuo to yield N-methyl-N-cyano3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 6. A solution ofpotassium hydroxide, water, ethylene glycol and of N-methyl-N-cyano3-(p-trifluoromethylphenoxy)-3-phenylpropylamine is placed in athree-neck, round-bottom flask equipped with magnetic stirrer andcondenser. The reaction mixture is heated to refluxing temperature for20 hours, and is then cooled. The reaction mixture is extracted withether. The ether extracts are combined, and the combined extracts washedwith water. The water wash is discarded. The ether solution is nextcontacted with 2 N aqueous hydrochloric acid. The acidic aqueous layeris separated. A second aqueous acidic extract with 2 N hydrochloric acidis made followed by three aqueous extracts and an extract with saturatedaqueous sodium chloride. The aqueous layers are all combined and madebasic with 5 N aqueous sodium hydroxide. N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 7, formed in the abovereaction, is insoluble in the basic solution and separated. The amine isextracted into ether. The ether extracts are combined, and the combinedextracts washed with saturated aqueous sodium chloride and then dried.Evaporation of the ether in vacuo yielded N-methyl3-(p-trifluoromethylphenoxy)-3-phenylpropylamine 7. Finally, thecompound 7 is then placed in DMF and activated with andO-(benzotriazol-1-yl)-N,N′,N′,N′-tetramethyluronium hexafluorophosphate(HBTU) and Diisopropylethylamine (DIEA). To the reaction mixture isadded 3-maleimidopropionic acid. The reaction is stirred for 3 hours.The organic phase is then washed with water and brine, dried over MgSO₄,chromatographied, triturated with cold ether and left to crystallizetoproduce 8 to produce the modified anti-depressant molecule

EXAMPLE 26 Maleimidopropionamyl-3,5-3′,5′ tetraiodothyroninamid(Modified-Thyroxine)

[0465] N-t-Boc-3,5-3′,5′ tetraiodothyronine 1 is placed in DMF andactivated with and O-(benzotriazol-1-yl)-N,N′,N′,N′,-tetramethyluroniumhexafluorophosphate (HBTU) and Diisopropylethylamine (DIEA) as indicatedin the schematic below. To the reaction mixture is added3-maleimidopropylamine. The reaction is stirred for 3 hours. The organicphase is then washed with water and brine, dried over MgSO₄, trituratedwith cold ether and left to crystallize to produce 2. Finally thecompound is placed in a 25% solution of TFA in CH₂Cl₂ for 15 minutes andthe CH₂Cl₂ is removed invacuo. The oily residue is then lyophilized toyield the desired compound 3, a modified thyroxine for treament ofthyroid deficiency, i.e., an anti-thyroid deficiency agent.

EXAMPLE 272S-hydroxy-3R-[1S-(MEEA-EDA-carbonyl)-2,2-dimethyl-propylcarbamoyl]-5-methylhexanohydroxamicacid (Modified MMPI)

[0466] The compound 1 and 3,4-dihydro-2H-pyran in CH₂Cl₂ and pyridiniump-toluenesulfornate are stirred at room temperature for 12 h asindicated in the schematic below. Then the solution is diluted withEtOAc and washed with half-saturated brine to remove the catalyst. Thesolvent is evaporated and the residue is treated with NaOH(1N) and EtOHfor 30 min. The solution is acidified with AcOH, and the product isextracted with EtOAc. The EtOAc solution is dried, evaporated to givethe THP ether 2. The compound 2, DCC and HOBT in CH₂Cl₂ are stirred atroom temperature for 60 min. Then MEEA-EDA HCl (N-(2-aminoethyl)[2-(2-maleiimidoethoxy)ethoxy]acetamide) and N-methylmorpholine areadded. The reaction is stirred for 2 h. and then quenched by addition ofAcOH. The precipitate is removed by filtration. The filtrate is washedwith diluted HCl, NaHCO₃ and dried. The crude product is used for thenext step.The crude product is treated with 2N HCl H₂O/EtOH 1:1) for 30min. EtOH is evaporated. The product is extracted with CH₂Cl₂. Thecombined CH₂Cl₂ layers are washed with NaHCO₃ and dried. Evaporation ofthe solvent gives a residue, which is purified by flash columnchromatography to afford 3. This compound can be purified further byHPLC on a reverse phase column and lyophilized to produce the modifiedMMPI.

EXAMPLE 28 Preparation of Rhodamine NHS Ester

[0467] Rhodamine Green™-X, succinimidyl ester, hydrochloride mixedisomers is commercially available from Molecular Probes (Eugene Oregon)as illustrated below:

EXAMPLE 29 In Vivo Addition of NHS-Rhodamine

[0468] New Zealand rabbits (2 Kg), male or female, were intramuscularlyanesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) andAcepromazine (0.75 mg/kg) prior to surgical exposure of left carotidartery. Both carotid arteries were isolated and blood flows weremeasured. A catheter (22G) was inserted in the arterial segment andrinsed with 0.9% sodium chloride via catheter until there was no morevisible evidence of blood in the segment.

[0469] A 1-cm incubation chamber was created by ligatures in the segmentarea. The incubation chamber was flushed three times with 1 mL of 0.9%sodium chloride. A solution of 100 μl of 500 μM NHS-Rhodamine wasprepared and incubated in the incubation chamber for 3 minutes. Theexcess of rhodamine was withdrawn with a 1 mL syringue. The incubationchamber was washed once again with 3 times 100 mL of 0.9% sodiumchloride. The incubation chamber was then removed from the rabbit, cutin three pieces and dipped in 10% formalin for further evaluation. TheNHS-Rhodamine treated arteries exhibited dramatic levels of fluorescencewhereas those arteries treated solely with Rhodamine exhibited littlefluorescence over background. These results demonstrate that Rhodaminewas covalently bonded to a local delivery site.

EXAMPLE 30 Preparation of [³H]-NHS-Propionate

[0470] [³H]-NHS-propionate is available from Amersham Canada Ltd.(Oakville, Ontario, Canada) and can be prepared from the tritiatedpropionic acid through known to the art condensation ofN-hydrosuccinimide in presence of EDC in DMF or methylene chloride.

EXAMPLE 31 In Vivo Pharmacokinetics Studies of [³H]-NHS-Propionate

[0471] New Zealand rabbits (2 kg), male or female, were intramuscularlyanesthetized with Xylazine (20 mg/kg), Ketamine (50 mg/kg) etAcepromazine (0.75 mg/kg) prior to surgical exposure of left carotidartery. Segments of 10 mm of carotids, were transiently isolated bytemporary ligatures and rinsed with 0.9% sodium chloride via a cannulauntil there was no more visible evidence of blood components.

[0472] A catheter (18G) was inserted in the arterial segment and servedto introduce the angioplasty balloon (2.5 mm of diameter, over thewire/Boston Scientific Inc.). A vascular damage (angioplasty) wasperformed on the isolated segment in order to eliminate the layer ofendothelial cells. The angioplasty balloon was serially inflated atdifferent atmospheres (4, 6, 8 and 10) during 1 minute, with 45 secondsof delay between inflations. At 4 atmospheres a balloon traction wasperformed 5 times and 1000 U/kg of heparin were infused in the bloodcirculation.

[0473] The angioplasty balloon was then retrieved from the artery andthe catheter was reintroduced. The arterial segment was rinsed 3 timeswith saline, and 100 μM of [³H]-NHS-propionate was incubated within theisolated segment of the artery for either 30 seconds, 3 minutes or 30minutes. At the end, the excess of incubation liquid was withdrawn fromthe artery, and the segment was rinsed 5 times with saline. The treatedartery was immediately harvested, and incorporation of [³H]-labeledcompounds within the artery was evaluated by scintillation counting.After 30 seconds of incubation, we recorded an association efficiency of2.55%. At 3 min and 30 min, we recorded an association efficiency of 5.5and 6.5%, respectively. We decided that a 3 min incubation time wassufficient to treat the artery in an efficient way.

[0474] When evaluating the retention levels, 100 μM of[³H]-NHS-propionate or [³H]-propionate were incubated with the arteryfor a period of 3 minutes, after which the segment has been rinsed 5times with saline. The catheter was then removed and the arteriotomysite was closed with microsutures, thus reestablishing the blood flowwithin the carotid. Finally, the neck wound was closed with sutures, andanimals are allowed to recuperate. Three days following the treatment,the animals are sacrificed with an overdose of sodium pentobarbital, thecarotid segments are removed and examined for compound's presence byscintillation counting. 10.94% retention of [³H]-NHS-propionate wasmonitored after three days following a 3 minute incubation period basedon residual radioactivity in the artery. The difference in retentionefficiency between covalently and non covalently bound propionate aftera 3 minutes incubation period was determined. An outstanding 12 foldenhancement in retention was recorded (0.6% of total amount incubatedagainst 0.046% for the non covalently bound) in favor of theNHS-propionate. This indicates that the tissue association of a compoundis dramatically enhanced by the covalent attachment in vivo. Subsequentrestitution of blood flow demonstrated retention [³H]-NHS-propionate ofapproximately 10% of the material 72 hours after injury. This representsexcessive tissue retention using the embodied technology of agentsmarkedly beyond that seen with all drug delivery technologies asexemplified in the literature for standard non covalent agents(Circulation 1994 89 (4) 1518-1524).

EXAMPLE 32 Synthesis of [³²P]NHS Derivative

[0475] To a solution of protected R and R′ (both R and R′) can be alkyl,phenyl or alkoxy groups, and X is either O or S, alkoxy, alkyl and anyother functionality stable under these conditions) phosphodiester (0.1mmol) and N-hydroxysuccinimide (0.2 mmol) is added diisopropylethylamine0.11 mmol), followed by addition of HBTU (0.22 mmol). The reactionmixture is stirred at room temperature for 36 hours. DMF is removed byvacuum distillation and the residue is dissolved in MeOH (10 mL). TheMeOH solution is filtered to remove the insolubles, the filtrate isconcentrated in vacuo, and the residue is dissolved in a minimum amountof MeOH. Water is then added to induce precipitation and the precipitateis dried on vacuum to give the desired compound.

[0476] The yield of the reaction can usually be improved by using EDC asthe coupling reagent, as exemplified below. To a solution of R and R′phosphodiester (0.054 mmol) and N-hydroxysuccinimide (0.115 mmol) inanhydrous DMF (3 mL), is added EDC (31 mg, 0.162 mmol). The solution isstirred at room temperature for 24 hours. DMF is removed by vacuumdistillation and the residue is further dried on high vacuum. Theresidue is then dissolved in a minimum amount of MeOH (0.12 mL) and H₂O(3.2 mL) is added to induce precipitation. The precipitates are washedwith H₂O (3×0.8 mL) and dried on vacuum to give a solid product.

[0477] Any protected phosphonate derivatives may undergo similartransformation.

EXAMPLE 33

[0478] New Zealand rabbits (2 kg), male or female, were anesthetizedwith xylazine (20 mg/kg), ketamine (50 mg/kg) and acepromazine (0.75mg/kg) intramuscularly prior to surgical exposure of left carotidartery. Carotid arteries were surgically dissected and segments ofapproximately 10 mm length were isolated. The vessels were cannulatedand rinsed with 0.9% sodium chloride until there was no more visibleevidence of blood components.

[0479] A catheter (18G) was inserted in the arterial segment and servedto introduce the angioplasty balloon (2.5 mm of diameter, over thewire/Boston Scientific Inc.). Vascular damage (angioplasty) wasperformed on the isolated segment in order to eliminate the layer ofendothelial cells. The angioplasty balloon was serially inflated atdifferent atmospheres (4, 6, 8 and 10) for 1 minute, with 45 seconds ofdelay between inflations. At 4 atmospheres a balloon traction wasperformed 5 times and 1000 U/kg of heparin were infused in the bloodcirculation.

[0480] The angioplasty balloon was then retrieved from the artery andthe catheter was reintroduced. The arterial segment was rinsed 3 timeswith saline, and 100 μM of [³²P]-NHS-[linking group] was incubatedwithin the isolated segment of the artery for 3 minutes. At the end, theexcess of incubation liquid was withdrawn from the artery, and thesegment was rinsed 5 times with saline. The vessel was sutured closed,blood flow restored and surgical wounds repaired. Animals were returnedto the vivarium for periods up to four weeks. Tissue retention of[³²P]-NHS-[linking group] was evaluating using whole animal radiographyat selected periods of time after injury.

EXAMPLE 34 Synthesis of [¹³¹I]-NHS Derivative

[0481] To a solution of protected amino protected [¹³¹I]-iodotyrosine(0.1 mmol) and N-hydroxysuccinimide (0.2 mmol) is addeddiisopropylethylamine (0.11 mmol), followed by addition of HBTU (0.22mmol). The reaction mixture is stirred at room temperature for 12 hours.DMF is removed by vacuum distillation and the residue is dissolved inMeOH (10 mL). The MeOH solution is filtered to remove the insolubles,the filtrate is concentrated in vacuo, and the residue is dissolved in aminimum amount of MeOH. Water is then added to induce precipitation andthe precipitate is dried on vacuum to give the desired compound.

[0482] The yield of the reaction can usually be improved by using EDC asthe coupling reagent, as exemplified below. To a solution of[¹³¹I]-iodotyrosine (0.054 mmol) and N-hydroxysuccinimide (0.115 mmol)in anhydrous DMF (3 mL), is added EDC (31 mg, 0.162 mmol). The solutionis stirred at room temperature for 24 hours. DMF is removed, by vacuumdistillation and the residue is further dried on high vacuum. Theresidue is then dissolved in a minimum amount of MeOH (0.12 mL) andwater (3.2 mL) is added to induce precipitation. The precipitates arewashed with H₂O (3×0.8 mL) and dried on vacuum to give a solid product.

EXAMPLE 35 In Vivo Pharmacology of ¹³¹I Derivative

[0483] New Zealand rabbits (2 Kg), male or female, were anesthetizedwith xylazine (20 mg/kg), ketamine (50 mg/kg) and acepromazine (0.75mg/kg) intramuscularly prior to surgical exposure of left carotidartery. Carotid arteries were surgically dissected and segments ofapproximately 10 mm length were isolated. The vessels were cannulatedand rinsed with 0.9% sodium chloride until there was no more visibleevidence of blood components.

[0484] A catheter (18G) was inserted in the arterial segment and servedto introduce the angioplasty balloon (2.5 mm of diameter, over thewire/Boston Scientific Inc.). Vascular damage (angioplasty) wasperformed on the isolated segment in order to eliminate the layer ofendothelial cells. The angioplasty balloon was serially inflated atdifferent atmospheres (4, 6, 8 and 10) for 1 minute, with 45 seconds ofdelay between inflations. At 4 atmospheres a balloon traction wasperformed 5 times and 1000 U/kg of heparin were infused in the bloodcirculation.

[0485] The angioplasty balloon was then retrieved from the artery andthe catheter was reintroduced. The arterial segment was rinsed 3 timeswith saline, and 100 μM of [¹³¹I]-NHS-[linking group] was incubatedwithin the isolated segment of the artery for 3 minutes. At the end, theexcess of incubation liquid was withdrawn from the artery, and thesegment was rinsed 5 times with saline. The vessel was sutured closed,blood flow restored and surgical wounds repaired. Animals were returnedto the vivarium for periods up to four weeks. Tissue retention of[¹³¹I]-NHS-[linking group] was evaluated using whole animal radiographyat selected periods of time after injury

EXAMPLE 36 Intrapulmonary Delivery of2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide.(Modified-Cetirizine)

[0486] A Bird Micronebulizer in line with a Bird Mark 7 respirator maybe charged with 5-10 ml of a solution of 12 mg/ml2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamidein mannitol/phosphate buffer. The Micronebulizer may then bes used tosimultaneously ventilate and dose a patient at 22 cm H₂O at a rate of1.8 mg/min for 30 min. At this pressure the patient shouldl ventilate atapproximately normal inspiratory volume. The patient should be allowedto exhale normally after each ventilated breath. In addition, thepatient should be positioned supine for dosing. After the first dosingperiod the pateint should be allowed to breathe normally for another 20minutes. After the 20 minute period, a second dosing should be performedin the same way as the first. Blood plasma samples should be taken atthe initiation time of the first dose and thereafter to monitor thelevels of2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide.

EXAMPLE 37 Intrapulmonary Delivery of2-[2-[4-[(4-chloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide(Modified-Cetirizine) Using the Spiros DPI System

[0487] The Spiros DPI is an aerosol generation system that is largelyindependent of the inspiratory flow rate and its use is described inU.S. Pat. No. 6,060,069.

[0488] A modified beclomethasone dipropionate (BDP) formulation may beprepared by first micronizing through conventional means (e.g., a jetmill) to produce a range of particle sizes that are likely to undergosedimentation in the human airway. Generally, fine particles in therange of 0.5 to 5.8 microns in diameter are thought to undergosedimentation between the oropharynx and small bronchioles. Particleswithin this general size category are thought to be in the “respirablerange.” Such micronized materials have excessive surface free energy,and as a result have a tendency to adhere strongly to many surfaces,most especially to themselves.

[0489] Lactose particles in the size range of 20 to 100 microns may bemixed with the smaller diameter micronized drug particles to create ahomogenous blend. Each lactose particle will generally bind to a numberof smaller drug particles in the blend. The blend flows more easilyduring the packaging and dose metering process.

[0490] The formulation may be then filled into cassettes, eachcontaining 30 individual doses. The cassettes may then packaged insealed foil pouches.

[0491] The following steps using the Spiros BPI system may be used todeliver a dose of inhaled drug: 1. The Spiros DPI does not need to beprimed; 2. The blue plastic cap is removed from the mouthpiece; 3. Theinhaler is held level; 4. The lid of the DPI is opened as far back aspossible (The lid will click when it has reached the correct angle); 5.The lid is then closed completely; 6. Before bringing the inhaler up tothe mouth, the patient breathes out, making sure not to breathe into theinhaler.; 7. The inhaler is brought up to the mouth in a level position;8. The lips are sealed fully around the mouthpiece, making sure there isno gap between the mouthpiece and the lips; 9. The patient breathes inthrough the mouth for about 4 seconds, preferably at a flow rate ofabout 20 LPM. The motor will turn on and the patient may taste/feel thedrug as it is inhaled; 10. The patient holds their breath for as long aspossible, up to 10 seconds. 11. The Spiros DPI is held in a levelposition during loading and dosing.

1 16 1 13 PRT Artificial Sequence Synthetic Peptide 1 Ala Gly Tyr LysPro Glu Gly Lys Arg Gly Asp Ala Lys 1 5 10 2 15 PRT Artificial SequenceSynthetic Peptide 2 Lys Arg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly ProPhe Cys 1 5 10 15 3 15 PRT Artificial Sequence Synthetic Peptide 3 LysArg Gly Asp Ala Cys Glu Gly Asp Ser Gly Gly Pro Phe Cys 1 5 10 15 4 36PRT Artificial Sequence Synthetic Peptide 4 Tyr Thr Ser Leu Ile His SerLeu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln GluLeu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 5 36PRT Artificial Sequence Synthetic Peptide 5 Tyr Thr Ser Leu Ile His SerLeu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln GluLeu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 6 36PRT Artificial Sequence Synthetic Peptide 6 Tyr Thr Ser Leu Ile His SerLeu Ile Glu Glu Ser Gln Asn Gln Gln 1 5 10 15 Glu Lys Asn Glu Gln GluLeu Leu Glu Leu Asp Lys Trp Ala Ser Leu 20 25 30 Trp Asn Trp Phe 35 7 36PRT Artificial Sequence Synthetic Peptide 7 Val Tyr Pro Ser Asp Glu TyrAsp Ala Ser Ile Ser Gln Val Asn Glu 1 5 10 15 Glu Ile Asn Gln Ala LeuAla Tyr Ile Arg Lys Ala Asp Glu Leu Leu 20 25 30 Glu Asn Val Lys 35 8 36PRT Artificial Sequence Synthetic Peptide 8 Lys Val Tyr Pro Ser Asp GluTyr Asp Ala Ser Ile Ser Gln Val Asn 1 5 10 15 Glu Glu Ile Asn Gln AlaLeu Ala Tyr Ile Arg Lys Ala Asp Glu Leu 20 25 30 Leu Glu Asn Val 35 9 35PRT Artificial Sequence Synthetic Peptide 9 Val Tyr Pro Ser Asp Glu TyrAsp Ala Ser Ile Ser Gln Val Asn Glu 1 5 10 15 Glu Ile Asn Gln Ala LeuAla Tyr Ile Arg Lys Ala Asp Glu Leu Leu 20 25 30 Glu Asn Val 35 10 31PRT Artificial Sequence Synthetic Peptide 10 His Ala Glu Gly Thr Phe ThrSer Asp Val Ser Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu PheIle Ala Trp Leu Val Lys Gly Arg Lys 20 25 30 11 30 PRT ArtificialSequence Synthetic Peptide 11 His Ala Glu Gly Thr Phe Thr Ser Asp ValSer Ser Tyr Leu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala TrpLeu Val Lys Gly Arg 20 25 30 12 8 PRT Artificial Sequence SyntheticPeptide 12 Pro Arg Lys Leu Tyr Asp Tyr Lys 1 5 13 23 PRT ArtificialSequence Synthetic Peptide 13 Arg Asn Pro Asp Gly Asp Val Gly Gly ProTrp Ala Trp Thr Thr Ala 1 5 10 15 Pro Arg Lys Leu Tyr Asp Tyr 20 14 12PRT Artificial Sequence Synthetic Peptide 14 Tyr Gly Arg Lys Lys Arg ArgGln Arg Arg Arg Lys 1 5 10 15 12 PRT Artificial Sequence SyntheticPeptide 15 Tyr Gly Arg Lys Lys Arg Arg Gln Arg Arg Arg Lys 1 5 10 16 13PRT Artificial Sequence Synthetic Peptide 16 Tyr Gly Gly Phe Leu Arg ArgIle Arg Pro Lys Leu Lys 1 5 10

We claim:
 1. A modified therapeutic agent comprising: a therapeuticagent and a reactive group which reacts in vivo with amino groups,hydroxyl groups or thiol groups on pulmonary components or bloodcomponents to form a stable covalent bond, the therapeutic agent beingselected from the group consisting of GP-41 peptides, BBB peptides,anti-cancer agents, antihistamines, bronchodilators, anti-hypertensionagents, anti-angina agents, opioids, analgesics, anti-depressants, andhypothyroid agents.
 2. The modified therapeutic agent of claim 1 whereinsaid reactive group is a succinimidyl or a maleimido group.
 3. Themodified therapeutic agent of claim 1 wherein said reactive group is amaleimido group which is reactive with a thiol group on a mobilepulmonary component.
 4. The modified therapeutic agent of claim 1wherein said reactive group is a maleimido group which is reactive witha thiol group on a fixed pulmonary component.
 5. The modifiedtherapeutic agent of claim 1 wherein said reactive group is a maleimidogroup which is reactive with a thiol group on a mobile blood component.6. The modified therapeutic agent of claim 1 wherein said reactive groupis a maleimido group which is reactive with a thiol group on albumin. 7.The modified therapeutic agent of claim 1 wherein said reactive group isa maleimido group which is reactive with a thiol group on a fixed bloodcomponent.
 8. The modified therapeutic agent of claim 1 wherein saidtherapeutic agent is an anti-histamine.
 9. The modified therapeuticagent of claim 1 wherein said therapeutic agent is a hypothyroid agent.10. The modified therapeutic agent of claim 9 wherein said therapeuticagent is loratidine.
 11. The modified therapeutic agent of claim 9wherein said therapeutic agent is cetirizine.
 12. An aerosol compositionfor delivery of a therapeutic agent to the pulmonary system of a hostcomprising: an aerosolized aqueous solution containing a modifiedtherapeutic agent, the modified therapeutic agent comprising atherapeutic agent and a reactive group which reacts with amino groups,hydroxyl groups or thiol groups on pulmonary or blood components to forma stable covalent bond.
 13. The aerosol of claim 12 further comprising apharmaceutically acceptable carrier.
 14. The aerosol of claim 12 whereinsaid modified therapeutic agent is 2.5-10% by weight.
 15. The aerosol ofclaim 12 wherein said therapeutic agent an anti-histamine.
 16. Theaerosol of claim 15 wherein said therapeutic agent is loratidine. 17.The aerosol of claim 15 wherein said therapeutic agent is cetirizine.18. A particulate formulation for delivery of a therapeutic agent to thepulmonary system of a host comprising: a dispersable dry powdercontaining a modified therapeutic agent, the modified therapeutic agentcomprising a therapeutic agent and a reactive group which reacts withamino groups, hydroxyl groups or thiol groups on pulmonary components toform a stable covalent bond.
 19. The particulate formulation of claim 18wherein at least 50% of the dry powder is in the form of particleshaving a diameter of 10 um or less.
 20. The particulate formulation ofclaim 18 wherein said therapeutic agent is an anti-histamine.
 21. Theparticulate formulation of claim 20 wherein said therapeutic agent isloratidine.
 22. The particulate formulation of claim 20 wherein saidtherapeutic agent is cetirizine.
 23. A method of delivering atherapeutic agent to a host comprising the steps of: obtaining amodified therapeutic agent, the modified therapeutic agent comprising atherapeutic agent and a reactive group which reacts in vivo with aminogroups, hydroxyl groups or thiol groups on pulmonary or blood componentsto form a stable covalent bond; and administering the modifiedtherapeutic agent to the pulmonary system of the host.
 24. The method ofclaim 23 wherein said administering step further comprises the steps ofaerosolizing the modified therapeutic agent for inhalation by the host.25. The method of claim 23 wherein said administering step furthercomprises the steps of dispersing a dry formulation of the modifiedtherapeutic agent for inhalation by the host.
 26. The method of claim 23wherein said administering step further comprises the steps ofinstilling the modified therapeutic agent into the pulmonary system ofthe host.
 27. The method of claim 23 wherein said reactive group is asuccinimidyl or a maleimido group.
 28. The method of claim 23 whereinsaid reactive group is a maleimido group which is reactive with a thiolgroup on a mobile pulmonary component.
 29. The method of claim 23wherein said reactive group is a maleimido group which is reactive witha thiol group on a fixed pulmonary component.
 30. The method of claim 23wherein said reactive group is a maleimido group which is reactive witha thiol group on a mobile blood component.
 31. The method of claim 23wherein said reactive group is a maleimido group which is reactive witha thiol group on a fixed blood component.
 32. The method of claim 23wherein said reactive group is a maleimido group which is reactive witha thiol group on human serum albumin.
 33. The method of claim 23 whereinsaid therapeutic agent is an anti-histamine.
 34. The method of claim 33wherein said therapeutic agent is loratidine.
 35. The method of claim 33wherein said therapeutic agent is cetirizine.
 36. Use of a compositionfor the manufacture of a medicament said composition comprising aderivative of an antihistamine and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananhistamine effect.
 37. Use of a composition according to claim 36wherein the antihistamine is selected from cetirizine, loratidine andanalogs thereof.
 38. Use of a composition according to claim 36 whereinthe antihistamine is selected from cetirizine and analogs thereof. 39.Use of a composition according to claim 36 wherein the antihistamine isselected from loratidine and analogs thereof.
 40. Use of a compositionfor the manufacture of a medicament said composition comprising aderivative of an anti-angina agent and analogs thereof wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananti-angina effect.
 41. Use of a composition according to claim 40wherein the anti-angina agent is tirofiban.
 42. Use of a composition forthe manufacture of a medicament said composition comprising a derivativeof an anti-hypertensive agent and analogs thereof wherein the derivativeincludes a reactive functional group which reacts with amino groups,hydroxyl groups, or thiol groups on blood components to form stablecovalent bonds, said reactive functional group being selected fromN-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimide groupfor use in the treatment of the human body to provide ananti-hypertensive effect.
 43. Use of a composition according to claim 42wherein the anti-hypetensive agent is enalapril.
 44. Use of acomposition for the manufacture of a medicament said compositioncomprising a derivative of an anti-arrhythmic agent and analogs thereofwherein the derivative includes a reactive functional group which reactswith amino groups, hydroxyl groups, or thiol groups on blood componentsto form stable covalent bonds, said reactive functional group beingselected from N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and amaleimide group for use in the treatment of the human body to provide ananti-arrhythmic effect.
 45. Use of a composition according to claim 44wherein the anti-arrhythmic agent is capobenic acid.
 46. Use of acomposition for the manufacture of a medicament said compositioncomprising a derivative of an anti-depressant agent and analogs thereofwherein the derivative includes a reactive functional group which reactswith amino groups, hydroxyl groups, or thiol groups on blood componentsto form stable covalent bonds, said reactive functional group beingselected from N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and amaleimide group for use in the treatment of the human body to provide ananti-depressan effect.
 47. Use of a composition according to claim 46wherein the anti-depressant agent is fluoxetine.
 48. Use of acomposition for the manufacture of a medicament said compositioncomprising a derivative of a bronchodilator and analogs thereof whereinthe derivative includes a reactive functional group which reacts withamino groups, hydroxyl groups, or thiol groups on blood components toform stable covalent bonds, said reactive functional group beingselected from N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and amaleimide group for use in the treatment of the human body to provide abronchodilation effect.
 49. Use of a composition according to claim 48wherein the bronchodilator is theobromineacetamine and analogs thereof.50. Use of a composition for the manufacture of a medicament saidcomposition comprising a derivative of an anti-inflammatory agent andanalogs thereof, wherein the derivative includes a reactive functionalgroup which reacts with amino groups, hydroxyl groups, or thiol groupson blood components to form stable covalent bonds, said reactivefunctional group being selected from N-hydroxysuccinimide,N-hydroxy-sulfosuccinimide and a maleimide group for use in thetreatment of the human body to provide an anti-inflammatory effect. 51.Use of a composition according to claim 50 wherein the anti-inflammatoryagent is loxoprofen and analogs thereof.
 52. Use of a composition forthe manufacture of a medicament said composition comprising a derivativeof an anti-thyroid deficiency agent and analogs thereof, wherein thederivative includes a reactive functional group which reacts with aminogroups, hydroxyl groups, or thiol groups on blood components to formstable covalent bonds, said reactive functional group being selectedfrom N-hydroxysuccinimide, N-hydroxy-sulfosuccinimide and a maleimidegroup for use in the treatment of the human body to provide ananti-thyroid deficiency effect.
 53. Use of a composition according toclaim 52 wherein the anti-thyroid deficiency agent is thyroxin andanalogs thereof.
 54. A composition comprising a compound selected fromthe group consisting of:2-[2-[4-[(4-choloropheny)phenylmethyl[-1-piperazinyl]ethoxy]-maleimidopropionylacetamide;11-(N-maleimidopropionyl-4-piperidylidene)-8-chloro-6,11-dihydro-5H-benzo-[5,6]-cyclohepta-[1,2-b]-pyridine;N-(1(S)-Ethoxycarbonyl-3-phenylpropyl)-L-alanyl-L-prolinylmaleimidopropionilamide;Maleimidopropynamyl-ε-(3,4,5-trimethoxybenz-amido)-caproicamide;Maleimidopropionamyl-1-theobromineacetamide;Maleimidopropamyl2-[4-(2-oxocyclopentan-1-ylmethyl)phenyl]propionamideN-maleimidopropionyl-N-methyl-3-(p-trifluoromethylphenoxy)-3-phenylpropylamine;4-anilino-1-(2-phenethyl)piperdine and Maleimidopropionamyl-3,5-3′,5′tetraiodothyroninamide.
 55. The composition of claim 54, wherein thecompound is Maleimidopropionamyl-3,5-3′,5′ tetraiodothyroninamide. 56.An aerosol composition for delivery of a therapeutic agent to thepulmonary system of a host comprising an aerosolized aqueous solutioncontaining a modified therapeutic agent conjugated to a blood protein.57. The composition of claim 56 wherein said protein is albumin.
 58. Theaerosol of claim 56 wherein said therapeutic agent an anti-histamine.59. The aerosol of claim 56 wherein said therapeutic agent isloratidine.
 60. The aerosol of claim 56 wherein said therapeutic agentis cetirizine.
 61. A particulate formulation for delivery of atherapeutic agent to the pulmonary system of a host comprising: adispersable dry powder containing a modified therapeutic agent, themodified therapeutic agent comprising a therapeutic agent and a reactivegroup which reacts with amino groups, hydroxyl groups or thiol groups onpulmonary components to form a stable covalent bond wherein saidtherapeutic agent is covalently bonded to a blood protein.
 62. Theformulation of claim 61 wherein said protein is albumin.
 63. Theformulation of claim 61 wherein said therapeutic agent is ananti-histamine.
 64. The formulation of claim 61 wherein said therapeuticagent is loratidine.
 65. The particulate formulation of claim 61 whereinsaid therapeutic agent is cetirizine.